BRPI0808673A2 - RECOMBINANT ANTIBODIES FOR TREATMENT OF RESPIRATORY SYNCHIAL VIRUS INFECTIONS. - Google Patents

Description

Report of the Invention Patent for "RECOMBINANT ANTIBODIES FOR TREATMENT OF RESPIRATORY SYNCHIAL VIRUS INFECTIONS".

FIELD OF THE INVENTION The present invention relates to antibody compositions by

and recombinant polyclonal, specific for the prevention, treatment or attenuation of one or more symptoms associated with respiratory syncytial virus infections. The invention also relates to polyclonal expression cell lines producing recombinant polyclonal antibody an10 ti-RSV (anti-RSV rpAb). Further, diagnostic and pharmacological compositions comprising anti-RSV rpAb and use in the prevention, treatment or attenuation of one or more symptoms associated with RSV infection are described.

BACKGROUND OF THE INVENTION Respiratory syncytial virus (RSV) is the leading cause of disease.

of the lower respiratory tract in infants and young children. Premature babies and children with underlying health problems, such as chronic lung disease or congenital heart disease, are at greatest risk for serious illness such as bronchiolitis and pneumonia following RSV infection. Recently, RSV has also been recognized as an important pathogen in certain high-risk adults, such as immunocompromised adults, particularly bone marrow transplant patients, the elderly, and individuals with chronic lung disease.

Human RSV is a member of the Pneumovirus 25 subfamily of the Paramyxoviridae family, and exists as a subtype AeB. RSV is an enveloped, non-segmented negative sense RNA virus. The viral genome encodes at least 11 proteins of which three are envelope-associated proteins, F (fusion glycoprotein), G (receptor binding glycoprotein), and SH (small hydrophobic protein). Envelope proteins are present on the viral surface, and to some extent on the surface of infected cells. Protein F promotes the fusion of viral and cellular membranes thus allowing the penetration of viral RNA into the cell cytoplasm. Protein F consists of two disulfide-linked subunits, Fi and F2, produced by proteolytic cleavage of an inactive, N-glycosylated precursor of 574 amino acids. Protein G is a 289-299 amino acid type II transmembrane glycoprotein (depending on the virus strain). The precursor form is 532 kDa, which matures to an 80-90 kDa protein by addition of both N- and O-linked oligosaccharides. RSV G protein is responsible for binding of virions to target cells. In addition to the membrane-bound form of protein G, a truncated, soluble form is also produced. It has also been suggested that its function is to redirect the immunoresponse away from the virus and infected cells. Thus, G protein has been shown to be associated with various proinflammatory effects such as modification of chemokine and cytokine expression as well as leukocyte recruitment. The SH protein is a 64-65 amino acid protein that is present in very small amounts on the surface of RSV peptides, but is abundantly expressed on the surface of RSV infected cells. The function of the SH protein has not been identified, but it may be able to aid the transport of virus protein through the Golgi complex (Rixon et al, 2002), J. Gen Virol. 85: 1153-1165). Blocking the function of GeF proteins is believed to be relevant for preventing RSV infection.

Prevention and treatment of RSV infection has received

Considerable attention over the past decades has included the development of vaccine, antiviral compounds (Ribavirin approved for treatment) antisense drugs, interference RNA technology (RNAi), and antibody products such as immunoglobulin and monoclonal antibodies (all 25 reviewed by Maggon and Barik, 2004, See. Med. Virol. 14: 149-168). Of these approaches, intravenous immunoglobulin RSV-IVIG and monoclonal antibody Palivizumab have been approved for RSV prophylaxis in high-risk children.

Immunoglobulin products such as RSV-IVIG (RespiGam) 1 are, however, known to have several disadvantages, such as low specific activity resulting in the need for large volume injection, which is difficult in children with limited venous access due to therapy. previous intensive. In addition, there is also a risk of transmission of viral diseases from serum-derived immunoglobulin products, as well as problems with variations from one batch to another. Finally, it is difficult to obtain donors that meet the needs for hyperimmune RSV immunoglobulin production, as only approximately 8% of normal donors have sufficiently high RSV neutralizing antibody titers.

Monoclonal antibodies to Fea protein G show that they have in vitro neutralizing effect and prophylactic effects in vivo (eg, Beeler and Coelingh 1989. J. Virol. 63: 2941-50; Garcia-Barreno et al. 1989. J. Virol 63: 925-32; Taylor et al., 1984. Immunology 52: 137-142; Walsh et al., 1984, Infection and Immunity 43: 756-758; US 5,842,307 and US 6,818,216). Palivizumab monoclonal antibody has now almost completely replaced the use of RSV-IVIG. Neutralization assays show that Palivizumab and RSV-IVIG perform equally well against RSV subtype B, while Palivizumab performs better against subtype A (Johnson et al. 1997. J. Infect. Dis. 176: 1215 -24). However, despite the good neutralizing and prophylactic effects of monoclonal antibodies as illustrated by products such as Palivizumab and Numax, they may also be associated with certain substances due to the nature of the RSV virus.

~ “RSV exists in two distinct antigenic groups or subtypes, A and B. Most RSV proteins are closely conserved between the two subgroups, with protein F showing 91% amino acid similarity. However, G protein exhibits extensive sequence variability, with only 53% amino acid similarity between AeB subgroups (Sullender 2000. Clin. Microbiol.Rev. 13: 1-15). Most proteins also show some limited intra-subgroup variation, except for G protein, which differs by up to 20% within subgroup A and 9% within subgroup B at the amino acid level. AeB virus subtypes co-circulate in most RSV epidemics, with the relative frequency varying between different years. Thus a monoclonal antibody must be carefully selected so that it is capable of neutralizing both subtypes as well as intra-subtype variations.

In addition to the issue of two RSV subtypes and intrasubtype diversity, human RSV, like most RNA1 viruses, has the ability to mutate rapidly under selective pressure. Selection of in vitro RSV escape mutants using mAb is well documented (e.g., Garcia-Barreno et al 1989. J. Virol. 63: 925-32). Importantly it was recently discovered that Palivizumab also selects escape mutants in vitro as well as in vivo, and that some of the isolated mutants are completely resistant to Palivizumab prophylaxis in cotton rats (Zhao and Sullender 2005. J.Virol. 79: 3962- 8 and Zhao et al., 2004. J.Infect.Dis. 190: 1941-6.). In addition, wild-type RSV strains that are intrinsically resistant to Palivizumab may also exist, as demonstrated by the murine antibody failure from which Palivizumab originates to neutralize a clinical isolate (Beeler and Coelingh 1989. J.Virol. 63 : 2941-50). In addition, an apparently resistant virus was also identified following Palivizumab prophylaxis in immunocompetent cotton rats (Johnson et al. 1997. J.Infect.Dis. 176: 1215-24). Thus, under certain conditions, the use of simple, monospecific antibody may not be adequate or sufficient for the treatment of RSV disease, as escape mutants exist or may develop over time as a result of treatment.

Another consideration regarding the utility of RSV-IVIG and Palivizumab is the dose required for effective treatment. 25 serum concentrations of more than 30 μg / mL have been shown to reduce pulmonary RSV replication 100-fold in the RSV-infected cotton rat model. For RSV-IVIG, a monthly dose of 750 mg intravenously administered total protein / kg was effective in reducing the incidence of hospitalization with RSV in high-risk children, whereas 30 for monthly intramuscular doses of Palivizumab of 15 mg / kg. kg proved to be effective. However, administration of high intravenous or intramuscular multiple doses is inconvenient for the patient, and prevents the product from being widely used for prophylaxis and treatment of the large group of adults at risk for RSV infection.

Thus, there is a need for an antibody product that is not dependent on donor availability, and which immunospecifically binds one or more RSV antigens spanning subtypes A and B, as well as any escape mutants due to virus mutations. is highly potent, has an improved pharmacokinetic profile, and thus has a total improved therapeutic profile, and therefore requires less frequent administration and / or lower dose administration.

Therefore, the object of the present invention is to provide a

A highly potent alternative recombinant anti-RSV immunoglobulin product that shows reactivity to respiratory syncytial virus subtypes A and B as well as to multiple epitopes on at least one of the major surface antigens to limit the possibility of escape mutations .

The invention is also intended to provide human anti-RSV antibody molecules, as well as derivatives thereof, wherein the antibody molecules or derivatives exhibit improved characteristics in reaction to monoclonal anti-RSV antibodies and antibody derivatives.

DESCRIPTION OF THE INVENTION

The invention relates to antibodies capable of competing in binding as antibody 824 as defined herein or with its Fab fragment. Antibody 824 binds poorly to recombinant protein, but with very little affinity to human RSV-infected cells, resulting in very powerful neutralization of RSV. In providing the 824 antibody, the inventors have identified an epitope that results in more effective in vitro and in vivo neutralization than seen before for any single RSV epitope. By providing antibody 824, the inventors also allowed the identification of other antibodies that bind to the same epitope. Such other antibodies may be of any origin and include binding fragments as well as affinity matured antibodies. Antibodies capable of competing with antibody 824 can be identified in a cell competition assay (determination of relative epitope specificities) as described in Example 1, section g-4. In other aspects, the invention relates to an anti-RSV antibody comprising CDRH3 having the following formula: CAX1X2X3X4X5X6PX7X8X9X10X11W,

Where Xi to Xn are individually selected from groups

from amino acids listed below X1 = R or K;

X 2 = D, E, N or Q;

X 3 = S, T, G or A;

X4 = S, T, G or A;

X5 = N, Q, D or E;

X6 = W, Y, F or H;

X7 = A, G, V, or S;

X8 = G, A, V, or S;

X9 = Y1F1WouH;

X-io = E or D; and Δ11 = D, E, N or Q; and a CDRL3 described by the formula: CXiX2X3X4X5X6PXyTF wherein Xi to X7 are individually selected from the amino acid groups listed below:

X1 = Q or H;

X 2 = Q, E or H;

X 3 = F, Y, W or H;

X4 = N, Q or H;

X5 = T, S, G or A;

X6 = Y, F, W or H; and X7 = F, Y, W or H.

Antibodies comprising such CDR sequences based on antibody 824 are expected to result in very effective virus neutralization both in vitro and in vivo as they are expected to bind to the same epitope. In other aspects, the invention relates to nucleic acids encoding such CDR1 sequences to nucleic acid encoding antibody 824 VL and VH sequences, expression vectors encoding such antibodies and CDRs, and cells expressing them.

Preferably, the anti-RSV antibody comprises the CDR1 and CDR2 regions of the 824 antibody VH and VL pair as shown in SEQ ID NOs: 232, 317, 487, and 572, and a CDRH3 region having the formula CARiD2S3S4N5W6PA7G8YgEioDiiW ( SEQ ID NO 402), and a CDRL3 region having the formula CQ1Q2F3N4T5Y6PF7TF (SEQ ID NO 657).

In another aspect, the invention relates to an antibody composition comprising an antibody based on the above CDR sequences and one or more additional anti-RSV antibodies.

The invention also relates to an antibody composition comprising distinct members comprising the heavy chain and light chain CDR1, CDR2 and CDR3 regions selected from the group of VH and VL pairs listed in Table 6, wherein the members The distinct members are the distinct members of one of the antibody compositions 2 through 56 in Table 9 herein.

In other aspects, the invention relates to a method of preventing, treating or alleviating one or more symptoms associated with an RSV infection in a mammal comprising administering an effective amount of an anti-RSV antibody according to the invention.

Use of a polyclonal antibody composition that targets epitopes in RSV is expected to minimize the development of escape mutants and may also provide against various naturally circulating viruses. In contrast to RSV-IVIG derived and sera, a poly25 clonal antibody of the present invention does not contain antibody molecules, which bind to non-RSV antigens.

The present invention provides a polyclonal anti-RSV antibody. Preferably, the polyclonal anti-RSV antibody is obtained from cells that do not naturally produce antibodies. Such an antibody is termed a recombinant polyclonal antibody (rpAb). An anti-RSV rpAb of the present invention is directed against multiple epitopes on protein F or G. In particular, an anti-RSV rpAb that is directed against multiple epitopes on both proteins. G and F is preferred. Preferably, the G protein epitopes belonging to the conserved group and also potentially the subtype specific group and strain specific group are covered by the anti-RSV rpAb. Additionally, antibodies with reactivity against the third envelope protein, small hydrophobic protein (SH) is a desired component of an anti-RSV rpAb of the present invention.

Further, the present invention provides pharmaceutical compositions wherein the active ingredient is an anti-RSV polyclonal antibody, as well as uses of such compositions for the prevention, attenuation or treatment of RSV infections.

The present invention further provides procedures for reflecting the humoral immunoresponse generated upon RSV infection by isolating the original Vh and VL gene pairs from such individuals and producing antibodies that maintain that original pairing.

Definitions

The term "antibody" describes a functional component of serum and is often referred to either as a collection of molecules (antibodies or immunoglobulin) or as a molecule (antibody molecule or immunoglobulin molecule). An antibody molecule is capable of binding to or reacting to a specific antigenic determinant (antigen or antigenic epitope), which in turn may lead to the induction of immune effector mechanisms. An individual antibody molecule is usually referred to as monospecific, and a composition of antibody molecules may be monoclonal (i.e. consisting of identical antibody molecules) or polyclonal (i.e. consisting of antibody molecules that react with them). different epitopes on the same or different, different antigens). Each antibody molecule has a unique structure that allows it to specifically bind to its corresponding antigen, and all natural antibody molecules have the same total basic structure of two identical light chains and two identical heavy chains. Antibodies are also collectively known as immunoglobulin. The terms antibody and antibodies as used herein are used in the broadest sense and include intact antibodies, chimeric, humanized, fully human and single chain antibodies, as well as antibody binding fragments such as Fab, Fv fragments or scFv fragments, as well as multimeric forms such as dimeric IgA or pentavalent 5 IgM molecules. In some instances, the present invention uses the term "synthetic or semi-synthetic antibody analog", which refers specifically to unnatural molecules that exhibit antibody characteristics (for exhibiting specific binding to RSV antigens) and includes natural antibody CDRs - such analogs are, for example, represented by 10 scFv fragments, unibodies, diabodies, etc., but could also be, for example, natural looking antibodies that are engineered to include CDRs (e.g., by grafting techniques known to the art. technique) of an anti-RSV antibody molecule described herein - for example, such an antibody analog could comprise CDRs described herein incorporated into an antibody molecule of another animal species or in a different antibody isotype or class of the same species. .

The term "recombinant anti-RSV polyclonal antibody" or "anti-RSV rpAb" describes a composition of recombinantly produced diverse antibody molecules, wherein the individual members are capable of binding to at least one epitope on a respiratory syncytial virus, and wherein the polyclonal composition as a whole is capable of neutralizing the -RSVrPreferably an anti-RSV rpAb composition neutralizes both RSV subtypes A and B. Even more preferred, the anti-RSV rpAb further comprises GeF protein binding reactivity. Preferably, the composition is produced from a single polyclonal cell line.

The term "Vh and VL encoder pair" describes an original pair of Vh and Vl decoding sequences contained within or derived from the same cell. Thus, the pair of Vh and Vl represents the pairing of Vh and Vl 30 present in the donor from which such a cell is derived. The term "an antibody expressed from a Vh and VL coding pair" indicates that an antibody or antibody fragment is produced from a vector, plasmid or the like containing the Vh and VL coding sequence. When a pair of Vh and Vl coders are expressed, either as a complete antibody or as a stable fragment thereof, it retains affinity for binding and specificity of the originally expressed antibody of the cell from which they are derived. 5 A library of cognate pairs is also called a repertoire or collection of cognate pairs, and can be held individually or in a meeting.

The term "a distinct member of a recombinant polyclonal antibody" denotes an individual antibody molecule of the composition of recombinant polyclonal antibody, comprising one or more extensions within the variable regions, which are characterized by differences in amino acid sequence compared to others. individual members of the polyclonal protein. These extensions are in particular located in the CDR1, CDR2, and CDR3 regions.

The term "epitope" is commonly used to describe a pro

portion of a larger molecule or part of a larger molecule (e.g., antigen or antigenic site) having antigenic or immunogenic activity in an animal, preferably a mammal, and more preferably in an epitope. An epitope having immunogenic activity is a portion of a larger molecule that elicits an antibody response in an animal. An epitope having antigenic activity is a portion of a larger molecule to which an antibody immunospecifically binds as determined by any method well known in the art, for example by the immunoassays described herein. Antigenic epitopes need not necessarily be immunogenic. An antigen is a substance to which an antibody or antibody fragment binds immunospecifically, for example, toxin, virus, bacteria, proteins or DNA. An antigen or antigenic site often has more than one epitope unless they are very small and is often capable of stimulating an immunoresponse. Antibodies 30 that bind to different epitopes on the same antigen may have varying effects on the activity of the antigen to which they bind depending on the location of the epitope. An antibody binding to an epitope at an active antigen site may completely block antigen function, while another antibody that binds to a different epitope may have no effect or little effect on antigen activity alone. Such antibodies may, however, still activate complement and thus result in antigen clearance, and may result in synergistic effects when combined with one or more antibodies that bind to different epitopes on the same antigen. In the present invention, the larger molecule of which the epitope is a ratio is preferably a ratio of an RSV polypeptide. The antigens of the present invention are preferably RSV-associated proteins, polypeptides or fragments thereof to which an antibody or antibody fragment binds immunospecifically. An RSV-associated antigen may also have an analog or derivative of an RSV polypeptide or fragment thereof to which an antibody or antibody fragment binds immunospecifically.

The term "entirely human being" used, for example, in re

DNA, RNA, or protein sequences describe sequences that are between 98 and 100% human.

The term "immunoglobulin" is commonly used as a collective designation of the antibody mixture found in blood or serum, but may also be used to denote an antibody mixture derived from other sources.

The term "reflects humoral immunoresponse" when used with respect to a polyclonal antibody refers to an antibody composition, wherein nucleic acid sequences encoding the members of individual antibodies are derived from a donor with an increased frequency of plasma cells that produce antiRSV specific antibodies. Such a donor may either be converted from an RSV infection, have had intimate contact with an RSV-infected individual, or have undergone RSV vaccination (for examples of RSV vaccines see, for example, Maggon and Barik, 2004, Rev Virol Med 14: 149-168). In order to reflect the affinity and specificity of antibodies produced in a donor upon infection or challenge, the sequences encoding the variable heavy chain (Vh) and the variable light chain (Vl) should be maintained in the pairs or combinations of genes originally present in the donor. donor (cognate pairs) when they are isolated. In order to reflect the diversity of a humoral immunoresponse in a donor, all sequences encoding RSV binding antibodies are selected based on the selection procedure. Isolated sequences are analyzed with respect to the diversity of variable regions, in particular the CDR regions, but also with respect to the Vh and VL family. Based on these analyzes, a population of cognate pairs representing the total diversity of the 10 RSV binding antibodies is selected. Such polyclonal antibody typically has at least 5, 10, 20, 30, 40, 50, 100, 1,000 or 104 distinct members.

A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient patient - the same naturally applies to excipients, vehicles and diluents that are part of a composition.

The term "polyclonal antibody" describes a composition of different (miscellaneous) antibody molecules that is capable of binding to or reacting with various specific antigenic determinants / epitopes on the same or different antigens, wherein each individual antibody in the composition is capable of reacting with a particular epitope. Usually, the variability of a polyclonal antibody is localized in the so-called variable regions of the polyclonal antibody, in particular in the CDR1, CDR2 and CDR3 regions. In the present invention, a polyclonal antibody may be produced either in a pot of a polyclonal cell line, or it may be a mixture of different polyclonal antibodies. A mixture of monoclonal antibodies is not as such considered a polyclonal antibody as they are produced in individual batches and not necessarily of the same cell line, which will result, for example, in post-translational modification differences. However, if a mixture of monoclonal antibodies provides the same antigen / epitope coverage as a polyclonal antibody of the present invention, it will be considered as an equivalent of the polyclonal antibody. When it is established that a member of a polyclonal antibody specifically binds or has specific reactivity against an antigen / antigen site / epitope, it is understood herein that the binding constant is below 100 nM. Preferably below 10nM, even more preferred below 1nM.

The term "recombinant antibody" is used to describe a

antibody molecule or molecules which are expressed from a cell or cell line transfected with an expression vector comprising the antibody coding sequence that is not naturally associated with the cell. If antibody molecules in a recombinant antibody composition are diverse or different, the term "recombinant polyclonal antibody" or "rpAb" applies according to the definition of a polyclonal antibody.

The term "recombinant polyclonal cell line" or "polyclonal cell line" refers to a mixture / population of cells expressing proteins that are transfected with a repertoire of variant nucleic acid sequences (for example, a repertoire of acid sequences). encoding nucleicides), which are not naturally associated with transfected cells. Preferably, the transfection is performed so that the individual cells, which together constitute the recombinant polyclonal cell line, each carry a transcriptionally active copy of a single nucleic acid sequence of interest encoding a member of the antibody. recombinant polyclonal of interest. Even more preferred, only a single copy of the distinct nucleic acid sequence is integrated into a specific site in the genome. The cells that make up the recombinant polyclonal cell line are selected for their ability to retain the integrated copy (or copy) of the nucleic acid sequence of interest, distinguished, for example, by antibiotic selection. Cells which may constitute such a polyclonal cell line may be, for example, bacteria, fungi, eukaryotic cells such as yeast, insect cells, plant cells or mammalian cells, especially immortal mammalian cell lines such as CHO cells. , COS cells, BHK cells, myeloma cells (e.g., Sp2 / 0 cells, NSO), NIH 3T3, YB2 / 0 and immortalized human cells such as HeLa cells, HEK 293 cells, or PER.C6.

The terms "sequences encoding Vh and VL" or "pairs encoding Vh and VL" indicate nucleic acid molecules, wherein each molecule comprises a sequence encoding the expression of a variable heavy chain and a light chain. variable, so that they can be expressed as a pair from the nucleic acid molecule if a suitable promoter and / or IRES regions are present and operably linked to the sequences. The nucleic acid molecule may also encode part of the full-length and / or light-chain constant or full-length constant regions, allowing expression of a Fab fragment, a life-size antibody or other antibody fragments, if suitable promoter. and / or IRES regions are linked or operably linked to the sequences.

A recombinant polyclonal antibody is said to be ad

administered in a "therapeutically effective amount" if the amount administered is physiologically significant, for example, prevents or attenuates an RSV infection in an animal or human being.

DESCRIPTION OF THE DRAWINGS Figure 1: (A) Alignment of amino acid sequences of the

Integral G protein from the prototype strains Long (subtype A) and 18537 (subtype B) The signal / transmembrane region is in a rectangle with dashed line. The two variable domains between amino acids 101-133 and 208-299 as identified by Cane et al. 1991 J. Gen. Virol. 72: 2091-2096 is dentified with an underscore. The central fragment of protein G has been expressed as a fusion protein in E. coli and is in a black rectangle. The 2 amino acid sequences are shown in SEQ ID NOs: 711- (subtype A) and 712 (subtype B). (B) Alignment of central fragment as indicated in (a). The location of the 13 aa conserved region (aa 164-176 residue) and the protein G cysteine-rich region (GCRR) are indicated in braces. GCRR disulfide bridges (identical for both subtypes) are indicated in square braces. The 2 amino acid sequences are shown in SEQ ID NOs: 713 (subtype A) and 714 (subtype B).

Figure 2: Multiplex overlap-extension RT-PCR scheme (A) and cloning steps (B). (A) Two primer groups, 5 CH + VH 1-8 and VK1-6 + CK1, specific for Vh and Vk gene families, respectively, were used for the first PCR step. A homologous region between the Vh and Vk primers results in the generation of an overlapping PCR product. In the second step, this product is amplified in the nested PCR. Primers also include restriction enzyme recognition sites that facilitate cloning. (B) The cognate linked Vh and VK coding pairs are assembled and inserted into a mammalian IgG expression vector (e.g., Figure 3) by use of the flanking Xho \ and Nott sites. Subsequently, a bidirectional promoter is inserted into the Asc \ -Nhe \ restriction site between the Vh and Vk coding sequences to facilitate expression of life-size antibodies. Used PCR primers are indicated by horizontal arrows. CH1: heavy chain constant domain 1, LC: constant domain, LC: light chain; Ab: antibody; P1-P2: bidirectional promoters.

Figure 3: Schematic presentation of a life size antibody expression vector 00-VP-530. The vector comprises the following elements: Amp and Amp pro = ampicillin resistant gene and its promoter. "PUC-" origin pUC origin of replication; P1 = mammalian promoter that triggers light chain expression. P2 - mammalian promoter that triggers heavy chain expression. IGHV leader = heavy chain, human, genomic leader. VH = heavy chain variable region coding sequence. IgGI = Sequence encoding the genomic immunoglobulin isotype G1 heavy chain constant region. Rabbit beta-globin poly-A sequence. Leader cap = sequence encoding light chain. Term SV40 = simian virus terminator sequence 40. FRT = Flp recognition target site. Neo = neomycin resistance gene. SV40 poly-A = Simian virus poly-A signal sequence 40.

Figure 4: Epitope specificity characterization of antibody obtained from clone 801 (Ab801) using Biacore analysis. 801 antibody binding was tested by peer competition for F1 protein binding using three antibodies, 9c5 (2), 133-h (3) and Palivizumab (4), which bind to antigenic sites F1, C and II, respectively. This reference cell illustrates non-competing Ab801 (1) protein F binding. Injection times of the four antibodies are indicated by an arrow. Response is indicated in units of relative resonance (UK). The long and double-headed arrows indicate the magnitude of the non-competing response and the double-headed arrow indicate the magnitude of the 9c5 inhibited response.

Figure 5: Shows results in in vitro neutralization of RSV strains of AeB subtypes. Dilutions of anti-F antibody mixtures were tested for their ability to neutralize RSV Long (Panel A) and RSV1 (Panel B) strains. The anti-F (1) antibody mixture obtained from clones 810, 818, 819, 825, and 827 is shown as triangles (A) and the anti-F (1) antibody mixture obtained from clones 735, 800 , 810, 818, 819, 825, 827, 863, 880, 884, and 894 is shown as squares (■). Palivizumab is shown as diamonds (♦), and a negative control antibody (anti-Rhesus D) corresponding to the isotype is shown as circles (·). Absorbance was measured at 490 nm and correlated with RSV replication.

Figure 6: Shows the results of an in vitro RSV fusion inhibition assay. Dilutions of antibody mixtures were tested for their ability to detect RSV BI. The anti-F (1) G antibody mixture obtained from clones 810, 818, 819, 825, 827, 793, 838, 841, 856 and 888 is shown as empty squares (□) and the antibody mixture, anti-F (ll) G, obtained from clones 735, 800, 810, 818, 819, 825, 827, 863, 880 , 884, 894, 793, 796, 838, 841, 856 and 888 is shown as empty triangles (Δ) Palivizumab is shown as diamonds (♦) Absorbance was measured at 490 nm and correlated with RSV.

Figure 7: Shows the results of an in vitro neutralization of RSV by combinations of anti-G antibody clones as measured by PRNT in the presence of active complement. Dilutions of individual antibody compositions (described in Table 9) were incubated with RSV Long strain in the presence of rabbit complement and thereafter left to infect Hep-2 cells. After 24 hours of incubation, the degree of infection was detected using RSV-specific plaque immunodetection. rpAb 13 anti-RSV is shown as empty triangles (Δ), rpAb 35 anti-RSV as triangles (A), rpAb 36 anti-RSV as squares (■), rpAB 41 anti-RSV as circles (·) and rpAb 4 anti -RSV as empty squares (□). Data are presented in% of infection compared to control ± SD; Figure 8. The pharmacokinetic profile of rpAb 33 and rpAb 56 anti-RSV in mice. BALB / c mice were treated with anti-RSV rpAb 33 and anti-RSV rpAb 56 (antibody compositions of Table 9) at a dose of 15 mg / kg. Serum samples were taken at various time points, ranging from day 0 (before inoculation) to day 29. Each point represents the mean serum human IgGI level at ± SD sampling time. DETAILED DESCRIPTION OF THE INVENTION Target antigens and polyclonal antibody compositions

A polyclonal antibody of the present invention is composed of several distinct antibody molecules in the same composition. Each molecule is selected based on its ability to bind to an RSV-associated antigen. A polyclonal antibody of the present invention comprises binding reactivity corresponding to the compiled binding reactivity of the distinct antibody molecules constituting the polyclonal antibody composition.

An anti-RSV polyclonal antibody of the present invention preferably comprises a binding reactivity compiled against both of the even more preferred GeFe proteins against multiple epitopes to minimize the risk of escape mutant development and obtain the greatest possible neutralization capability. At least five major antigenic sites that are recognized by neutralizing antibodies have been identified in protein F (Lopez et al 1988. J. Virol. 72: 6922-8). All antigenic sites have been mapped to the F1 chain, and include sites I, II, IV, V1, and VI, where the Iell sites may also be called BeA, respectively. Site II is located in a protease-resistant region in the N-terminal segment, and sites IV, V, and V1 at the C-terminal end of the protein's cysteine-rich region. Site I is located in the cysteine group. Another antigenic site on protein F is site C in which the F2 epitope including amino acid positions 241 and 242 is located. Additionally, there are monoclonal antibodies that bind to an antigenic site called F1, comprising epitopes named F1a, F1b and F1c. Currently, this antigenic site has not been mapped to a particular site on protein F, but these appear to overlap with site I. Most of these sites / epitopes give rise to broadly neutralizing antibodies, 10 but some antibodies to antigenic site I have been shown. be specific for subtype A. Antibodies that bind to site I also have marginal effect on virus neutralization. The epitope recognized by Palivizumab is located at the II antigen site as judged by the location of the selected escape mutations at amino acid position 272 (Zhao et al. 15 2004. J.Infects.Dis. 190: 1941-6). In addition, three types of epitopes were identified in the G protein: i) conserved epitopes that are present in all strains of RSV, ii) group-specific epitopes that are in all viruses belonging to only a subset of strains belong to the same subtype. . Conserved and group-specific epitopes were mapped to the central part of protein G containing a group of four cysteines (amino acid residue 173, 176, 182 and 186) and a short amino acid segment (residues 164-176) of sequence. identical among all human RSV isolates. The cysteine group is maintained by disulfide bonds between position 173-183 and 176-182 and forms the central part of the protein G cysteine-rich region (GCRR) ranging from amino acid residue 171-187, so that GCRR overlaps with the conserved 13 amino acid region. Glycoprotein G appears to play a role in both inducing protective immunity and disease pathogenesis. For example, mouse studies have shown that G-protein30 primers for a CD4 + Th2 T cell response are characterized by the production of IL-4, IL-5, IL-13 and pulmonary eosinophilia. Recruitment and activation of eosinophils are promoted by several factors, such as IL-4 and IL-5. In addition, RSV G protein expression during acute mouse infection has been associated with a modified innate immunoresponse, characterized by reduced expression of Th1 cytokine (eg, lL-2 and gamma-interferon), chemokine mRNA alteration expression (e.g. 5 MIP-1 alpha, MIP-1 beta, MIP-2, IP-10, MCP-1), and NK cell trafficking in modulating the innate inflammatory response, thus potentially delaying RSV renewal. (Polack et al. 2005. PNAS 102: 8996-9001). GCRR comprises a CX3C motif at amino acid positions 182 to 186. Reduction in respiratory rates in RSV 10 infected mice has been shown to be associated with the CX3C motif, as antibodies against this motif abolish the reduction in respiratory rates (Tripp et al. 2003 J. Virol 77: 6580-6584 and US 2004/0009177 (Application 10 / 420.387)). The strain-specific epitopes are preferably located at the third C-terminal variable of polypeptide G, although a strain-specific epitope has been mapped to an N-terminal variable region for the G protein ectodomain cysteine group (Martinez et al 1997. J. Gen. Virol. 78: 2419-29). Figure 1 shows an alignment of Long strain G (subtype A) and 18537 (subtype B) G proteins, indicating the various regions of protein G. Generally, monoclonal anti-protein G antibodies have marginal effects on the neutralization of the protein. RSV. However, mixtures of monoclonal anti-G antibodies have been reported to enhance RSV neutralization in vitro as well as in vivo (Walsh et al. 1999). J.Gen.Virol. 70: 2953-61 and Martinez and Melero 1989 J.Gen.Virol. 79: 2215-20). The greatest effect of combining monoclonal anti-G antibodies is apparently obtained when antibodies 25 bind to different epitopes, although a fraction of the virus still remains resistant to neutralization. Additionally. Combinations of two different anti-F antibodies with different epitope specificities as well as combinations of an anti-F and an anti-G antibody have been shown to have an enhanced in vitro neutralizing effect on RSV (Anderson 30 et al. 1988. J. Virol 62: 4232-4238). Some of the advantages obtained by mixing monoclonal antibodies appear to be due to the individual properties of monoclonal antibodies, such as an antagonistic effect, for example by blocking the active site. Other effects appear to be synergistic for reasons that are not currently understood.

The mechanisms of neutralization of RSV are complex and not completely understood. The large number of different epitopes, conserved subtype-specific as well as strain-specific epitopes identified on FeG proteins alone, as well as the potential generation of escape mutants suggests that a broad spectrum of antibody specificities is needed to target all neutralization mechanisms that may play a role in preventing infection with 10 RSV. Thus, it would be very difficult to rationally select the mixture of monoclonal antibodies capable of preventing RSV infection with RSV strain of both AeB subtypes, as well as escape mutants and new strains arising from the known RSV strains today.

One aspect of the present invention is to provide a polyclonal anti-RSV antibody with considerable diversity and broad anti-RSV specificity. The polyclonal anti-RSV antibody of the present invention is not dependent on donor availability during production and the batch-to-batch variation is considerably less than that observed for donor-derived anti-RSV immunoglobulin products (e.g., RSV IVIG). . In a polyclonal anti-RSV antibody of the present invention all members of the individual antibodies are capable of binding to the RSV-associated unrrantigen and the polyclonal antibody is capable of neutralizing RSV A and B subtypes. It is preferred that each antibody other than the polyclonal antibody binds to an epitope that is not bound by any of the other members of the polyclonal antibody. A polyclonal anti-RSV antibody of the present invention will bind to RSV antigens in a multivalent manner, which usually results in synergistic neutralization, phagocytosis of macrophage-infected cells, and enhanced antibody-dependent cellular cytotoxicity (ADCC) against infected cells as well as activation of increased complement. In addition, a polyclonal antibody of the present invention is not "diluted" by non-binding protein which is the case for RSV IVIG, where a dose of 750 mg total protein / kg is required to be effective. The percentage of RSV-specific antibodies within 750 mg of total protein, but is unlikely to constitute more than at most 1%, and most likely less. Thus, when the in vitro potency of Palivizumab has been estimated to be 25-30 times greater than 5 than that of RSV IVIG (Johnson et al. 1997, J. Infect.Dis. 176: 1215-24), this is the tradeoff. reduced RSV IVIG specific activity. Thus, if only 1% of the immunoglobulin molecules contained in RSV IVIG are specific for RSV, then the active dose of RSV IVIG polyclonal antibody is only 7.5 mg, which is lower than that of Palivizumab monoclonal antibody.

For these reasons, a recombinant polyclonal RSV-specific antibody of the present invention is expected to be significantly more potent than a monoclonal antibody, and therefore it will be possible to administer a lower dose of a polyclonal antibody of the present invention compared to those of the present invention. effective doses of Palivizumab and IVIG of RSV. Thus, a polyclonal anti-RSV antibody of the present invention is also considered suitable for the prophylaxis and treatment of high-risk adults, in particular bone marrow transplant recipients, elderly individuals and individuals with chronic lung disease. Another advantage of a polyclonal anti-RSV antibody of the present invention is that the concentration of the individual antibody members is significantly lower than the concentration of a monoclonal antibody (even if the dose used is the same), the possibility that an antibody individual is recognized as foreign by the immune system of the individual being treated is decreased, even if an individual antibody is eliminated by an immunoresponse in the patient, this cannot affect the neutralizing capacity or rate of renewal of the polyclonal anti-RSV antibody. as the antibody members remain intact.

One embodiment of the present invention is a recombinant polyclonal anti-RSV antibody capable of neutralizing RSV subtypes A and B, and when said polyclonal antibody comprises distinct antibody members that specifically bind to at least three different epitopes on at least one RSV envelope protein. Preferably, protein F is specifically bound by at least three distinct antibody members, and said epitopes are preferably located at different antigenic sites.

Another embodiment of the present invention is an antibody

recombinant polyclonal ti-RSV capable of neutralizing RSV subtypes A and B, and wherein said polyclonal antibody comprises distinct antibody members which together provide specific reactivity against at least two RSV envelope proteins. The two envelope proteins may be selected from RSV G protein, RSV F protein and RSV SH protein. Preferably, the polyclonal anti-RSV antibody of the present invention comprises anti-G and anti-F reactivity. The anti-G and anti-F reactivity of such polyclonal antibody is preferably comprised of at least two distinct anti-G antibodies and at least one distinct anti-F antibody. Preferably, at least three distinct antibodies bind to different epitopes, thus encompassing at least three different epitopes, and together the antibodies are capable of equally neutralizing the RSV subtype A and subtype strains. Even more preferred, the anti-G and anti-F reactivity of a polyclonal anti-RSV antibody of the present invention is comprised of any combination of the anti-G and anti-F reactivity described below. More preferably, a polyclonal anti-RSV antibody of the present invention is comprised of anti-G and anti-F reactivity against all antigenic sites / epitopes mentioned below. To obtain the possible specificity of a polyclonal anti-RSV antibody of the present invention, it is desired that one or more, preferably all antigenic sites are covered by more than one distinct antibody. Accordingly, it is preferred that several epitopes on the same antigen or antigenic site are linked by distinct members of the polyclonal unTantibody of the present invention.

With respect to the anti-G reactivity of a polyclonal anti-RSV antibody of the present invention, this reactivity is preferably directed against conserved epitopes. Even more preferred, anti-G reactivity is comprised of a first anti-G antibody capable of specifically binding to a conserved G protein epitope, and a second antiG antibody capable of specifically binding to the cysteine rich region of protein G ( GCRR). The anti-RSV antibody preferably comprises at least two distinct anti-G antibodies, wherein at least one first antibody is capable of specifically binding to a conserved G protein epitope, and at least one second antibody is capable of specifically binding. to a different conserved epitope or a group-specific epitope that recognizes both subtype A and subtype B. Preferably, the polyclonal antibody comprises at least three distinct anti-G antibodies wherein the first antibody is capable of specifically binding to an epitope conserved on protein G, and the second antibody is capable of specifically binding to subtype A protein G and the third antibody is capable of specifically binding to subtype B protein G. The cysteine-rich region of protein G (GCRR) specifically overlaps the upstream 13 amino acid region, where conserved epitopes are located, and a region where specific epitopes are of the group are located. Thus, antibodies capable of binding specifically to a conserved epitope as well as group-specific antibodies may be binding to GCRR if the epitope they recognize is located in GCRR. Preferably, at least one of the distinct antibodies characterized by their binding to a conserved epitope or a strain-specific epitope also recognizes GCRR. Antibodies that bind to the CX3C d GCRR motif are especially preferred from a virus neutralization point of view. However, antibodies that bind to CX3c motifs can also bind to several other unrelated human antigens, such as fractalcin and other human CX3C chemokines, and thus produce unwanted side effects meaning that it will be a rational approach to test such antibodies for cross-reactivity. (e.g. as demonstrated for certain antibodies in the examples) and later test the same antibodies in suitable model systems. At any rate it will always be necessary to test a given pharmaceutical product, such as an antibody of the present invention, in a clinical experiment before it can be established with a degree of certainty that side effects are absent, minor or at least acceptable. In addition to conservative and group-specific anti-G reactivity, anti-G reactivity directed against strain-specific epitopes can also be comprised in the polyclonal anti-RSV antibody of the present invention. Strain-specific anti-G reactivity directed against the abundant strain-specific epitopes present in virus strains that resulted from RSV infection within the last five years is preferred. In the present invention, strain-specific epitopes are understood to be epitopes that are only present in a limited number of RSV strains. Addition of group-specific and / or strain-specific anti-G antibodies may provide additional diversity to an anti-RSV antibody of the present invention, and may induce synergy when in combination with antibodies with reactivity to the conserved G protein region. The anti-G antibodies of the present invention directly neutralize RSV, block virus entry into the cell, prevent cell migration, inhibit inflammatory responses, and / or prevent syncytia formation.

With respect to the anti-F reactivity of a polyclonal anti-RSV antibody of the present invention, this reactivity is preferably directed against at least one epitope at one or more of the I, II, IV, V, VI, C or F1 antigenic sites. In other embodiments of the present invention, at least two, three, four, five, six or all of these antigenic / epitope sites are encompassed by the distinct antibodies in a polyclonal anti-RSV antibody of the present invention. Preferably, the anti-F antibodies of the present invention directly neutralize RSV and / or block virus entry into the cell and / or prevent syncytia formation.

In polyclonal anti-RSV antibody compositions of the present invention wherein the composition does not comprise binding reactivity directed against all antigenic sites on protein F, the presence of at least one distinct anti-F antibody that specifically binds to an epitope of the preferred antigenic site II. Even more preferred, the site-specific anti-F1 antibody binds to the same epitope or antigenic site as the Palivizumab antibody. In addition to site-specific antibodies, one or more distinct anti-site-specific anti-F antibodies are desired, such antibody preferably binds to the same epitope as RSVF2-5.

Subtype-specific anti-F antibodies are also known in the art. However, since protein F shows 91% amino acid similarity between the two subgroups A and B, subtype-specific anti-F antibodies are less abundant than for anti-G antibodies. Such strain-specific anti-F antibodies will, however, contribute to obtaining as broad a specificity as possible, and thus are also desired components of a polyclonal anti-RSV antibody of the present invention.

In addition to the RSV G and F protein antigens mentioned above, the RS virus expresses a third envelope protein, small hydrophobic protein (SH). Hyperimmune sera produced against SH protein peptides have been shown to be unable to neutralize RSV 15 in vitro (Akerlin-Stopner et al. 1993 J. Med. Virol. 40: 112-120). However, since the protein is primarily expressed in infected cells, we believe antibodies against the SH protein will have an effect on fusion inhibition and are potentially relevant for in vivo protection against RSV infections. This is based on the fact that RSV strains that fall from the SH gene replicate 10 times less efficiently in the upper respiratory tract (Bukreyev et al. 1997 J. Virol. 71: 8973-82).

A further embodiment of the present invention is an anti-RSV antibody capable of neutralizing RSV AeB subtypes and comprising anti-SH reactivity, and anti-G or anti-F reactivity. The C-terminus ranging from amino acid 41 to 64/65 (A / B subtype) of the SH protein is exposed on the cell surface. Thus, anti-SH reactivity against an epitope located in this area is desired. The C-terminus of the SH protein ranges from A and B subtypes, and it is therefore desired to include anti-SH reactivity against both A and B subtypes in a polyclonal antibody of the present invention. Such SH reactivity may be provided by at least two distinct antiSH antibodies where the first antibody is capable of specifically binding to the SH subtype A and the second antibody is capable of specifically binding to the SH subtype B.

In one embodiment of the present invention, a polyclonal antiRSV antibody comprises specific reactivity against SH subtypes A and / or B as well as specific reactivity against protein G. The reactivity against protein G may be composed of any of the above mentioned reactivity.

In an alternative embodiment, the specific reactivity against SH subtypes A and / or B may be combined with any of the anti-F reactivity described above to constitute a polyclonal anti-RSV antibody.

In a preferred embodiment of the present invention, a polyclonal anti-RSV antibody comprises reactivity against all three envelope proteins, F, G and SH.

The reactivity comprised in a polyclonal anti-RSV 15 antibody of the present invention may constitute any possible combination of distinct antibodies with specific binding reactivity against the antigens / antigenic sites and / or epitopes summarized in Table 1, provided that the combination is capable of neutralizing RSV subtypes A and B. Preferably, the combination contains reactivity against at least two RSV envelope proteins.

Preferably, members of the individual distinct antibody of a polyclonal antibody according to the present invention have neutralizing and / or anti-inflammatory properties of their own. Antibodies without these particular properties may, however, also play a role in RSV renewal, for example by complement activation. Antigen Antigenic Site / Epitope Protein F Antigenic Site I Antigenic Site Il Antigenic Site IV Antigenic Site V Antigenic Site Vl Antigenic Site C Epitope F1 Protein G Conserved Region (aa 164-176) Subtype A Specific B Subtype B GCRR (aa 171- 187) (conserved as well as strain specific) Reason CX3C (aa 182-186) Strain specific SH Protein Subtype A Subtype B Preferably, a polyclonal antibody of the present invention is

It is produced as a single batch or several batches of a polyclonal cell line that is not naturally expressing antibody molecules (also referred to as recombinant polyclonal antibody expression designation). One of the advantages of producing a recombinant polyclonal antibody compared to a monoclonal antibody mixture is the ability to produce an unlimited number of distinct antibody molecules at the same time (and at a cost similar to producing a single monoclonal antibody). Thus, it is possible to include antibodies reactive to a large number of RSV-associated antigens without significantly increasing the cost of the final product. In particular, with such a complex target as RSV, of which biology is not fully understood, individual antibodies that have not been shown to neutralize or protect against RSV alone can, when combined with other antibodies, induce a synergistic effect. Thus, it may be advantageous to include distinct antibodies, other than those described above, in a polyclonal antibody composition, in which the sole criterion is that the individual antibody binds to an RSV-associated antigen (e.g., assessed by binding to cells infected with 5 RSV). Preferably, all of the polyclonal anti-RSV antibody compositions described above are recombinant polyclonal anti-RSV antibody (rpAB) compositions.

One way to acquire potentially relevant antibodies that bind to the RSV target antigens, which have not been found to be relevant but may be, is to generate a polyclonal antibody composition that is composed of individual antibodies produced by a immunoresponse from a donor who was infected with RSV (complete immunoresponse). In addition to obtaining antibodies that represent a complete RSV immunoresponse, positive selection for antibodies that bind to antigens that are likely to be of particular relevance in protecting, neutralizing, and / or eliminating RSV infections can be performed. Further, if antibodies to a particular antigen, antigenic site or epitope believed to be of relevance in the protection, neutralization and / or elimination of RSV are not identified in the complete donor immunoresponse, such antibodies may be produced by immunization / vaccination of a donor with that particular antigen (selected immunoresponders). Generally, neutralization is evaluated by in vitro neutralization assays such as plaque reduction, micro neutralization or fusion inhibition assays (e.g., Johnson et al. 1997. J.Infect.Dis. 176: 1215-24 Thus, an antibody or antibody composition having a significant effect in one of the assays, when compared to a negative control, is considered to be neutralizing. Protection is generally assessed by in vivo challenge experiments, for example in the rat model. cotton (e.g., Johnson et al. 1997. J. Infect. Dis. 176: 1215-24) or the murine model (eg Taylor et al. 1984. Immunolgy 52, 137-142 and Mejias, et al. Antimicrob Chemother Agents 49: 4700-4707) In vivo challenge experiments can be performed either in a prophylactic mode, where antibodies are administered prior to the viral challenge or as a treatment in which antibodies are administered after viral challenge or as a combination of both the.

A polyclonal antibody composition of the present invention may be composed of antibodies capable of binding to an RSV antigen that is not necessarily known or not necessarily an envelope protein (the antibody binds to infected cells, but not to selected antigens or sites antibodies), but when antibodies are acquired from a complete immunoresponse following RSV infection, for example by obtaining nucleic acid sequences encoding antibodies distinct from one or more RSV-infected donors or recovering from RSV infection. Second, antibodies from the same complete immunoresponse that have been selected based on their ability to bind to a particular antigen, antigenic site and / or epitope can be included in the polyclonal antibody of the present invention. Third, distinct Vh and Vl pair-encoded antibodies obtained and one or more donors that have been immunized / vaccinated with a particular RSV-related antigen, thereby producing a "selected" immunoresponse in those donors, may be included in an antibody composition. polyclonal compounds of the present invention. Thus, antibodies derived from any of the techniques mentioned in the present invention may be combined into a single polyclonal antibody. Preferably, the nucleic acids encoding the antibodies of the present invention are obtained from human donors and the antibodies produced are entirely human antibodies.

The motivation behind the polyclonal antibody compositions of the present invention is: If an RSV-infected donor produces a humoral immunoresponse against an antigen, those antibodies are likely to at least to some extent contribute to viral turnover and thus qualify. for inclusion in a polyclonal antibody product.

Another aspect of the present invention is to produce an antiRSV rpAB wherein the composition of the distinct antibody members mirrors the humoral immunoresponse with respect to the diversity, affinity and specificity against RSV envelope antigens. Preferably, humoral response mirroring is established by ensuring that one or more of the following are filled in: i) nucleic acid sequences encoding the Vh and Vl regions of individual antibody members in such antiRSV rpAB are donor-derived ( (s) that have produced a humoral immunoresponse against RSV, for example following RSV infection; ii) the Vh and Vl coding sequences are isolated from the donor (s) so that the original matching of the Vh and Vl 10 coding sequences present in the donor (s) is maintained, iii) the Vh and VL pairs encoding the individual rpAB members are selected such that the CDR regions are as diverse as possible, or iv) the specificity of the individual members of the anti-RSV rpAb are selected such that the antibody composition It binds collectively to antigens that elicit significant antibody responses in mammals. Preferably, the antibody composition binds collectively to antigens, antigenic sites and / or epitopes that produce significant antibody titers in a serum sample from said donor (s). Such antigens, antigenic sites and / or epitopes are summarized in Table 1 above, but may also consist of unknown antigens, antigenic sites and / or epitopes as well as non-envelope antigens as described above. Preferably, the donors are human, and the polyclonal antibody is an entirely human antibody.

The present invention has identified a series of Vh and Vl 25 pairs that can be expressed as full length antibodies, Fab fragment or other antibody fragments that have binding specificity for an RSV-associated antigen. Specific Vh and Vl pairs are identified by the number of clones in Table 6 of Example 2. An antibody containing a pair of Vh and VL as identified in Table 6 is preferably a life-size antibody. However, if desired, chimeric antibodies may be produced as well.

A preferred recombinant anti-RSV polyclonal antibody of the present invention is composed of distinct members comprising heavy chain and light chain CDR1, CDR2 and CDR3 regions selected from the Vh and Vl pair group listed in Table 6. Preferably, CDR regions are maintained in the pairing indicated in Table 6 and inserted into a desired structure. Alternatively, the heavy chain CDR regions (CDRH) of a first clone are combined with the light chain CDR regions (CRDL) of a second clone (mixing Vh and VL pairs). CDR regions may also be mixed within the light chain or heavy chain, for example by combining the CDRL1 region of a first clone with CDRL2 region and CDRL3 region of a second clone. Such mixing is preferably performed among clones that bind to the same antigen. The CDR regions of the present invention may also be affinity matured, for example by point mutations.

Preferred Antibody Compositions

Particularly preferred antibody compositions comprising more than one distinct antibody molecule have been identified by the present inventors. These include antibody compositions that are effective in virus (Table 9) and in vivo (Table 10 and 11) neutralization assays.

A particularly preferred antibody composition is an antibody composition comprising antibody 824 or an antibody derived therefrom as described herein and one or more additional anti-RSV antibodies. Antibody 824 itself is a very potent antibody and when combined with other antibodies - particularly antibodies directed against protein G - exhibit very high potency in vitro and in vivo.

The one or more additional anti-RSV antibodies may be selected from the group consisting of human antibodies, humanized antibodies, and chimeric mouse antibodies.

Preferably, the one or more additional anti-RSV antibodies are selected from the group consisting of the antibody molecules listed in Table 6 herein, or specifically a binding fragment of said antibody molecule or a semi-synthetic antibody analog, wherein said fragment. The binding or analogue moiety comprises at least the complementarity determining regions (CDRs) of said isolated antibody molecule 5, except for an antibody having clone 824 CDRs.

A preferred antibody composition is composed of distinct members comprising heavy chain and light chain CDR1, CDR2, and CDR3 regions selected from the Vh and Vl pair group listed in Table 6, wherein the distinct members are the distinct members of a single chain. 10 of antibody compositions 2 to 56 in Table 9 below. Such antibody compositions have been shown to be potent in neutralizing one or more RSV strains in vitro.

Preferably, the antibody composition is capable of neutralizing the RSV subtype A in a virus neutralization assay, more preferably the composition is also capable of neutralizing the RSV subtype A in a virus neutralization assay, more preferably the composition. It is also capable of neutralizing the RSV B subtype in a virus neutralization assay. Suitable assays include plaque reduction and microneutralization assays described herein.

Particularly preferred compositions have been shown to

in vivo and comprise the distinct members selected from the distinct members of one of the antibody compositions 2, 9, 13, 17, 18, 28, 33, and 56 of Table 9 below.

In some embodiments, the antibody composition does not contain anti-RSV antibodies other than the distinct limb CDR antibodies described for each composition 2-56 of Table 9 below. Isolation and Selection of Variable Heavy Chain and Variable Light Chain Coding Pairs

The process of generating a recombinant polyclonal anti-RSV antibody composition involves isolating the sequences that encode variable heavy chains (Vh) and variable light chains (Vl) from a suitable source, thereby generating a repertoire of Vh and VL coding pairs. . Generally, a suitable source for obtaining Vh and Vl coding sequences is cell fractions containing lymphocytes such as blood, spleen and bone marrow samples from an animal or human infected with RSV or recovering from an infection. with RSV, or 5 from an animal or human immunized / vaccinated with an RSV strain or from proteins or DNA derived from such strain. Preferably, lymphocyte-containing fractions are collected from humans or from transgenic animals with human immunoglobulin genes. The collected lymphocyte-containing cell fraction may be further enriched to obtain a particular lymphocyte population, for example, B-lymphocyte lineage cells. Preferably, enrichment is performed using magnetic bead cell classification (MACS) and / or fluorescence activated cell classification (FACS), taking advantage of lineage-specific cell surface marker proteins, for example, B-cells, plasma blasts and / or plasma cells. Preferably, the fraction of cells containing lymphocytes is enriched with respect to B cells, plasma blasts and / or plasma cells. Even more preferred, cells with high expression of CD37 and intermediate expression of CD19 and / or CD45 are isolated from blood. These cells are sometimes called circulating plasma cells, early plasma cells, or plasma blasts. For convenience, these are readily referred to as plasma cells in the present invention, although other terms may be used interchangeably.

Isolation of coding sequences can be or performed in the classical manner, wherein the Vh and Vl coding sequences are randomly combined into a vector to generate a combinatorial library of Vh and Vl coding sequences. However, in the present invention , it is reported to mirror the affinity diversity and specificity of antibodies produced in a humoral immunoresponse upon infection with 30 RSV. This involves maintaining the pairing of Vh and Vl originally present in the donor, thus generating a repertoire of sequence pairs where each pair encodes a variable heavy chain (Vh) and a variable light chain (Vl) corresponding to a pair of sequences. Vh and Vl originally present in an antibody produced by the donor from which the sequences are isolated. This is also called a cognate pair of sequences encoding Vh and Vl and the antibody is called a cognate antibody. Preferably, the combinatorial or cognate Vh and Vl coding pairs of the present invention are obtained from human donors, and therefore the sequences are completely human.

There are several different approaches to generating cognate pairs of Vh and VL coding sequences; one approach involves amplifying and isolating Vh and Vl coding sequences from single cells chosen from a cell fraction containing lymphocytes. Vh and Vl coding sequences can be separately amplified and paired in a second step or can be paired during amplification (Coronella et al. 2000, Nucleic Acids Res. 28: E85; Babcook et al. 1996. PNAS 93: 7843 -7848 and WO 2005/042774). A second approach involves cell amplification and pairing of Vh and Vl coding sequences (Embleton et al. 1992. Nucleie Acids Res. 20: 3831-3837; Chapai et al. 1997. BioTechniques 23: 518-524). A third approach is the selected lymphocyte antibody (SLAM) method which combines a hemolytic plaque assay with Vh and Vl cDNA cloning (Babcook et al. 1996. PNAS 93: 7843-7848). As a repertoire of Vh and Vl encoding sequence pairs resembling the diversity of Vh and Vl sequence pairs in the donor, a high throughput method with minimal mixing (random combination) of the Vh and Vl pairs is preferred. See, for example, as described in WO 2005/042774 (incorporated herein by reference).

In a preferred embodiment of the present invention, a repertoire of Vh and VL coding pairs, wherein the pairs of limbs mirror the gene pairs responsible for the humoral immunoresponse resulting from RSV infection, is generated according to a method comprising steps i) providing a lymphocyte-containing cell fraction from an RSV infected donor or recovering from an RSV infection; ii) optionally enriching B cells or plasma cells from said cell fraction; iii) obtaining an isolated single cell population, comprising distributing the cells from said cell fraction individually in a plurality of vessels; iv) amplifying and ligating the Vh and VL coding pairs in a multiplex overlap-extension RT-PCR procedure and v) optionally nesting the linked Vh and Vl coding pairs. Preferably, the cognate pairs encoding Vh and Vl are subjected to a selection procedure as described below.

After the sequence pairs of Vh and Vl are generated, it is performed

a selection procedure to identify sequences encoding pairs of Vh and Vl with antigen binding reactivity associated with an RSV. Preferably, the RSV-associated antigen is an RSV envelope protein, in particular the RSV G protein, RESV F protein, and RSV SH protein15. If the Vh and Vl sequence pairs are combinatorial, a phage exposure procedure can be applied to enrich the Vh and Vl pairs that encode RSV-binding antibody fragments prior to selection.

In order to mirror the diversity, affinity and specificity of antibodies produced in a humoral immunoresponse upon RSV infection, the present invention has developed a selection procedure for cognate pairs to achieve the widest possible diversity. For selection purposes, the repertoire of Vh and Vl cognate pairs is individually expressed either as antibody fragments (eg scFv or Fab) or as life-size antibodies using either bacterial or mammalian selection vector in a suitable host cell. . The Fabs / antibody repertoire is selected for reactivity in a suitable host cell. The Fabs / antibody repertoire is selected for virus particle reactivity of one or more RSV strains. Preferably, at least two strains, one of subtype A and one of subtype B are used. Strains of subtype A are, for example, Long (ATCC VR-26), A2 (ATCC VR-1540) or later or subtype A isolates such as Longer. Subtype A strains are, for example, 18537 (ATCC VR1580), B1 (ATCC VR-1400), 9320 (ATCC VR-955) or newer isolates such as 18537. In parallel, the repertoire of Fabs / antibodies is selected against antigens. such as recombinant G protein, recombinant 5 F protein and RSV antigen derived peptides. Antigenic peptides may be, for example, selected from the conserved G protein region (amino acids 164-176) and cysteine nucleus region (amino acid 171-187 of the G protein subtype A and subtype B strains) and from the G protein region. SH extracellular protein (amino acids 42-64 of subtype A and 42-6510 of subtype A). Preferably, the peptides are biotinylated to facilitate immobilization on beads or plaques during selection. Alternative immobilization means may be used as well. Antigens are selected based on knowledge of RSV biology and the expected neutralizing and / or protective effect that antibodies capable of binding to these antigens may potentially provide. This selection procedure may also be applied to a combinatorial library of phage exposure. Recombinant F and / or F proteins used for selection may be expressed in bacteria, insect cells or another expression system. G and / or F proteins may be either expressed as a soluble protein (without the transmembrane region) or they may be fused to a

I

third protein to increase stability. If G and / or F proteins are expressed with a fusion marker, the fusion partner may be cleaved prior to selection. Preferably, the G and / or F proteins representative of both the A and subtype B subtypes are expressed and used for selection. Additionally, strain-specific G proteins may be expressed and used for selection. In addition to the primary selection described above, a secondary selection may be performed to ensure that none of the selected sequences encodes false positives. In the second selection, all RSV / antigen binding Vh and Vl pairs identified in the first selection are reselected against both the selected virus strains and antigens. Generally, immunological assays are suitable for the selection made in the present invention. Such assays are well known in the art and constitute, for example, ELISPOTS, ELISA, FLISA, membrane assays (e.g., Western blots), filter series, and FACS. The assays may be or performed without any previous enrichment using polypeptides produced from the Vh and VL pairs coding sequences. In the case that the repertoire of Vh and Vl coding pairs are cognate pairs, no enrichment, for example by phage exposure, is required prior to solution. However, in the selection of combinatorial libraries, immunoassays are preferably performed in combination with or following enrichment methods such as phage exposure, ribosome exposure, bacterial surface exposure, yeast exposure, eukaryotic virus exposure, RNA exposure. or covalent exposure (reviewed by FitzGeraId, K., 2000. Drug Discov. Today 5, 253-258).

The sequences encoding Vh and Vl pairs selected in the selection are generally subjected to sequencing, and analyzed with respect to the diversity of variable regions. In particular, diversity in the CDR regions is of interest, but also the representation of the VH and V1 family is of interest. Based on these analyzes, sequences encoding the Vh and Vl pairs that represent the total diversity of RSV binding antibodies isolated from one or more donors are selected. Preferably, sequences with differences in all CDR regions (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2 and CDRL3) are selected. If there are sequences with one or more identical or very similar CDR regions that belong to different families of Vh or Vl, these are selected as well. Preferably, at least the variable heavy chain CDR3 region (CDRH3) differs among the selected sequence pairs. Potentially, the selection of sequence pairs of Vh and Vl may be based only on the variability of the CDRH3 region. During sequence initiation and amplification, mutations may occur in the framework regions of the variable region, in particular in the first framework region. Preferably, errors that occur in the first framework region are corrected to ensure that the sequences match completely or at least 98% of those of the donor, for example, so that the sequences are entirely human.

When it is ensured that the total diversity of the collection of selected sequences encoding Vh and Vl pairs is highly representative of the diversity seen at the genetic level in a humoral response to an RSV infection, it is expected that the full specificity of antibodies expressed from of a collection of selected Vh and Vl coding pairs is representative with respect to the specificity of antibodies produced in RSV-infected donors. An indication of whether

I

The specificity of antibodies expressed from a collection of Vh and V1 coding pairs is representative of the specificity of antibodies produced by infected donors can be obtained by comparing antibody titers with respect to virus strains as well as selected blood donor antigens. the specificity of antibodies expressed from 15 a collection of Vh and VL coding pairs. Additionally, the specificity of antibodies expressed from a collection of Vh and Vl coding pairs can be analyzed subsequently. The degree of specificity correlates with several different antigens whose binding reactivity can be detected. In another embodiment of the present invention, the specificity of the individual antibodies expressed from a collection of Vh and Vl coding pairs is analyzed by epitope mapping.

Epitope mapping can be performed by various methodologies, which do not necessarily exclude each other. One way to map the epitope specificity of an antibody clone is to assess binding to peptides of varying lengths derived from the primary structure of the target antigen. Such peptides may both be linear and conformational and may be used in various assay formats, including ELISA1 ELISA and surface plasmon resonance (SPR, Biacore). In addition, peptides may be rationally selected using sequence and structure data to represent, for example, extracellular regions and conserved target antigen regions, or they may be drawn as a panel of overlapping peptides depicting a selected portion. or the entire antigen (Meloen RH, Puijk WC1 Schaaper WMM. Epitope mapping by PEPSCAN. In: Immunology Methods Manural. Ed Iwan Lefkovits 1997, Academic Press, pp. 982-988). Specific reactivity of an antibody clone with one or more such peptides will generally be an indication of epitope specificity. However, peptides are in many cases poor mimetics of epitopes recognized by antibodies raised against protein antigens, both due to lack of conformation and due to the generally larger surface area covered by interaction between an antibody and a protein antigen compared to an antibody and a peptide. A second method for epitope mapping, which allows the definition of specificities directly on the protein antigen, is by selective epitope masking using existing well-defined antibodies. Reduced binding of a second probe antibody to the antigen following blockade is generally indicative of shared or overlapping epitopes. Epitope mapping by selective masking can be performed by a variety of immunoassays, including, but not limited to, ELISA and Biacore1 which are well known in the art (e.g., Ditzel et al. 1997. J. Mol. Biol. 267: 684-695 Aldaz-Carroll et al 2005. J. Virol 79: 6260-6271). Still another potential method for determining the epitope specificity of antivirus antibodies is the selection of viral escape mutants in the presence of antibody. Sequencing of the disinterest gene (s) of such escape mutants will generally reveal which amino acids in the antigen (s) that are important for antibody recognition and thus constitute (part) of the epitope.

Preferably, individual members to be comprised of an anti-RSV rpAb of the present invention are selected such that the specificity of the antibody composition collectively encompasses both RSV subtypes A and B, as well as RSV associated antigens, proteins F and G, and preferably also SH.

Production of a recombinant polyclonal antibody from Vh and Vl coding pairs

A polyclonal antibody of the present invention is produced from a polyclonal expression cell line in one or a few reactors or equivalents thereof. Following this approach, anti-RSV rpAb can be purified from the reactor as a simple preparation without having to separate the individual members that make up anti-RSV 5 rpAb during the process. If the polyclonal antibody is produced in more than one bioreactor, the supernatants from each bioreactor may be pooled prior to purification, or the purified anti-RSV rpAb may be obtained by pooling the antibodies obtained from individually purified supernatants from each bioreactor.

One way of producing a recombinant polyclonal antibody is

described in WO 2004/061104 and WO 2006/007850 (PCT / DK2005 / 000501) (these documents are incorporated herein by reference). The method described therein is based on site-specific integration of the antibody coding sequence into the individual host cell genome, ensuring that the Vh and Vl protein chains are maintained in their original pairing during production. In addition, site-specific integration minimizes "position effects" and therefore the growth and expression properties of individual cells in the polyclonal cell line are expected to be very similar. The method generally involves the following: (i) a host cell with one or more recombinase recognition sites, (ii) an expression vector with at least one recombinase recognition site compatible with that of the host cell: (iii) generation of a collection of expression vectors by transfer of the Vh and Vl encoding pairs of the selection vector for an expression vector so that a full-size antibody or fragment thereof can be expressed from the vector (such transfer may not be necessary if the selection vector is identical to the vector). iv) transfection of the host cell with the collection of expression vectors and a vector encoding a recombinase capable of combining the recombinase recognition sites in the host cell genome with that in the vector; v) obtaining / generating a transfected host cell polyclonal cell line; and vi) expressing and collecting the polyclonal antibody from the host cell line.

Preferably, mammalian cells such as CHO1 cells, COS cells, BHK cells, myeloma cells (e.g., Sp2 / 0 or NSO cells), fibroblasts such as NIH 3T3, and immortalized human cells such as HeLa cell, HEK cells are used. 293, or PER.C6. However, non-mammalian or prokaryotic eukaryotic cells, such as plant cells, insect cells, yeast cells, fungi, E. coli, etc., may also be employed. A suitable host cell comprises one or more suitable recombinase recognition sites in its name. The host cell must also contain a selection mode that is operably linked to the integration site in order to be able to select integrants, (i.e. cells having an integrated copy of an anti-RSV Ab expression vector or vector fragment of expression at the integration site). Preparation of cells having an FRT site at a predetermined site terminated in the genome has been described, for example, in US Patent 5,677,177. Preferably, a host cell has a simple integrating site, which is located at a site that allows high expression of the integrant (the so-called hot spot).

A suitable expression vector comprises a recombination recognition site corresponding to the host cell recombinase recognition site (s). Preferably, the recombinant recognition site is linked to a suitable selection gene other than the selection gene used for the construction of the host cell. Selection genes are well known in the art, and include glutamine synthetase (GS) gene, dihydrofolate reductase gene (DHFR), and neomycin, where GS DHFR can be used for Vh and Vh sequence gene amplification. Vl inserted. The vector may also contain two different recombinase recognition sites to allow recombinase-mediated cassette exchange (RMCE) of the antibody coding sequence rather than complete integration of the vector. RMCE is described by Langer et al 2002. Nucleic Acids Res. 30, 3067-3077; Schlake and Bode 1994. Biochemistry 33, 12746-12751 and Belteki et al 2003. Nat. Biotech. 21, 321- 324. Suitable recombinase recognition sites are well known in the art, and include FRT1 Iox and attP / attB sites. Preferably, the integrating vector is an isotype-encoding vector, where constant regions (preferably including introns) are present in the vector before transferring the Vh and Vl coding pair from the selection vector (or constant regions are already present in the vector). selection vector if selection is performed on life-size antibodies). The constant regions present in the vector can be either the entire heavy chain constant region (CH1 to CH3 or CH4) or the constant region encoding the Fc portion of the antibody (CH2 to CH3 or CH4). The light chain Capa or Lambda constant region may also be present prior to transfer. The choice of the number of constant regions present, if any, depends on the selection or transfer system used. Heavy chain constant regions can be selected from the IgGI, IgG2, IgG3, IgG4, IgAI, IgA2, IgM1 IgD and IgE isotypes. Preferred isotypes are IgGI and / or IgG3. In addition, the expression vector for site-specific integration of the anti-RSV antibody-encoding nucleic acid contains suitable promoters or equivalent sequences that direct the high expression levels of each of the Vh and V1 chains. Figure 3 illustrates one possible way of drawing the expression vector, although numerous other drawings are possible.

The transfer of the Vh and Vl coding pairs from the selection vector may be accomplished by conventional restriction enzyme cleavage and ligation such that each expression vector molecule contains a Vh and VL coding pair. Preferably, the coding pairs of Vh 25 and Vl are individually transferred, however, they may also be bulk transferred if desired. When all selected Vh and Vl coding pairs are transferred to the expression vector, a collection or expression vector library is obtained. Alternative modes of transfer may also be used if desired. If the selection vector 30 is identical to the expression vector, the expression vector library is made up of the sequence pairs of Vh and Vl selected during selection which are situated in the selection / expression vector. Methods for transfecting a nucleic acid sequence into a host cell are known in the art. To ensure site-specific interaction, a suitable recombinase should be provided to the host cell as well. This is preferably obtained by co-transfecting a recombinase encoding plasmid. Suitable recombinants are, for example, Flp, Cre or phage OC31 integrase, used together with a host cell / vector system with recombinase recognition sites. The host cell may be either mass transfected, meaning that the expression vector library is transfected into cell line 10 in a single reaction thereby obtaining a polyclonal cell line. Alternatively, the expression vector collection may be transfected individually into the host cell, thereby generating a collection of individual cell lines (each cell line produces an antibody with a particular specificity). Transfection-generated cell lines (individual or polyclonal) are then selected for site-specific integrants, and adapted to grow in suspension and serum-free media if they no longer possess these properties prior to transfection. If transfection was performed individually, individual cell lines are further analyzed for their growth and antibody production properties. Preferably, cell lines with similar proliferation rates and antibody expression levels are selected for generation of the polyclonal cell line. The polyclonal cell line is then generated by mixing the individual cell lines at a predefined ratio. Generally, a polyclonal master cell bank (pMCB), a polyclonal research cell bank (pRCB) and / or a polyclonal work cell bank (pWCB) is deposited over the polyclonal cell line. Polyclonal cell line is generated by mixing individual cell lines at a predefined ratio. The polyclonal cell line is distributed into ampoules, thus generating a polyclonal research cell bank (pRCB) or a master cell bank (pMCB) from which a working cell bank (pWCB) can be generated by expansion. from the search cell bank or master. The research cell bank is primarily for proof of concept studies, in which the polyclonal cell line cannot comprise as many individual antibodies as the polyclonal cell line in the master cell bank. Typically, 5 pMCB is expanded to subsequently produce a pWCB for production purposes. Once the pWCB has run out, a new pMCB ampoule can be expanded to formulate a new pWCB.

One embodiment of the present invention is a polyclonal cell line capable of expressing a recombinant polyclonal anti-RSV antibody of the present invention.

Another embodiment of the present invention is a polyclonal cell line, wherein each individual cell is capable of expressing a pair of single Vh and Vl coders, and the polyclonal cell line as a whole is capable of expressing a collection of Vh and 15 VL coding pairs, wherein each pair of Vh and Vl encode an anti-RSV antibody. Preferably, the collection of coding pairs of Vh and Vl are cognate pairs generated according to the methods of the present invention.

A recombinant polyclonal antibody of the present invention is expressed by culturing an ampoule of a pWCB in an appropriate medium for a period of time that permits sufficient antibody expression and wherein the polyclonal cell line remains stable. days to 50 days). Culture methods such as fed batch or infusion may be used. Recombinant polyclonal antibody is obtained from the culture medium and purified by conventional purification techniques. Affinity chromatography combined with subsequent purification steps such as ion exchange chromatography, hydrophobic interactions and gel filtration have often been used for IgG purification. After purification, the presence of all individual members in the polyclonal antibody composition is assessed, for example, by ion exchange chromatography. The characterization of a polyclonal antibody composition is described in detail in WO 2006/007853 (PCT / DK2005 / 000504) (incorporated herein by reference). An alternative method for expressing a mixture of antibodies in a recombinant host is described in WO 2004/009618. This method produces antibodies with different heavy chains associated with the same light chain from a single cell line. This approach may apply if anti-RSV rpAb is produced from a combinatorial library.

Therapeutic Compositions

Another aspect of the invention is a pharmaceutical composition comprising as an active ingredient an anti-RSV rpAB or a recombinant polyclonal anti-RSV Gab 10 or a recombinant polyclonal anti-RSV antibody fragment. Preferably, the active ingredient of such a composition is a recombinant polyclonal anti-RSV antibody as described in the present invention. Such compositions are intended for the prevention and / or treatment of RSV infections. Preferably, the pharmaceutical composition is administered to a human, domestic animal, or pet.

The pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Anti-RSV rpAb or polyclonal fragments thereof may be administered in a pharmaceutically acceptable diluent, carrier or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide formulations or compositions suitable for administration to RSV-infected patients, or to patients who may be at high risk of RSV-infected. In a preferred embodiment, administration is therapeutic, meaning that it is administered after the onset of symptoms related to RSV infection. Any appropriate route of administration may be employed, for example, administration may be parenteral, intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, intranasal, aerosol, suppository, or oral administration. For example, pharmaceutical formulations may be in the form of tablets, capsules, chewing gum or confection, and for intranasal formulations, in the form of powders, nasal drops or aerosols.

The pharmaceutical compositions of the present invention are prepared in a manner known per se, for example by means of conventional dissolving, lyophilizing, mixing, granulating or confectioning processes. Pharmaceutical compositions may be formulated according to conventional pharmacy practice (see, for example, Remington: The Science and Practice of Pharmacy (20th Edition), ed. AR Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia, PA and Encyclopeda of Pharmaceutical Technology, eds J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York, NY).

Preferably, solutions or suspensions of the active ingredient, and especially isotonic aqueous solutions or suspensions, are used in the preparation of the pharmaceutical compositions of the present invention. In the case of lyophilized compositions comprising the active ingredient alone or together with a carrier, for example mannitol, such solutions may, if possible, be produced prior to use. The pharmaceutical compositions may be sterile and / or may comprise excipients, for example preservatives, stabilizers, wetting and / or emulsifying agents, solubilizers, osmotic pressure regulating salts and / or buffers, and are prepared in a manner known per se, for example by means of conventional dissolution or lyophilization processes. Said solutions or suspensions may comprise viscosity enhancing substances such as sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatin.

Injection compositions are prepared in the usual manner under sterile conditions; The same also applies to introducing the compositions into ampoules or vials and sealing the containers.

Pharmaceutical compositions for oral administration may be obtained by combining the active ingredients with solid carriers, if desired, granulating the resulting mixture and processing the mixture, if desired or necessary, after addition of the appropriate excipients, 30 tablets, pills, or capsules, which may be coated with gum, sugar or both. It is also possible for these to be incorporated into plastic vehicles which allow the active ingredients to diffuse or to be released in metered amounts.

The pharmaceutical compositions comprise from about 1% to about 95%, preferably from about 20% to about 90% active ingredient. The pharmaceutical compositions according to the invention may be, for example, in unit dose form, such as ampoules, vials, suppositories, tablets, pills, or capsules. The formulations may be administered to human subjects in therapeutically or prophylactically effective amounts (e.g., amounts that prevent, eliminate or reduce a pathological condition) to provide therapy for a disease or conditions. The preferred dosage of the therapeutic agent to be administered is likely to depend on such variables as the severity of the RSV infection, the general health of the particular patient, the formulation of the excipients of the compounds and their route of administration. of the compositions according to the invention.

The pharmaceutical compositions according to the present invention may be used for the treatment, attenuation or prophylaxis of a disease in a mammal. Conditions that may be treated or prevented with the present pharmaceutical compositions include prevention, and treatment of patients infected with RSV, or at risk of becoming infected with RSV, in particular patients who may be at high risk of becoming infected with RSV. High-risk patients are, for example, babies and young children. In particular, premature babies and children with an underlying problem such as chronic lung disease or congenital heart disease are at high risk of developing serious diseases such as bronchiolitis and pneumonia following RSV infection. Also high-risk adults, such as immunocompromised adults, particularly recipients of elderly bone marrow transplantation and individuals with chronic lung disease, may preferably undergo prophylactic or therapeutic treatment with a pharmaceutical composition according to the present invention.

One embodiment of the present invention is a method for preventing, treating or alleviating one or more symptoms associated with RSV infection in a mammal, comprising administering an effective amount of a recombinant anti-RSV polyclonal antibody of the present invention to said mammal.

Another embodiment of the present invention is the use of an

The recombinant polyclonal antibody of the present invention in the preparation of a composition for the treatment, attenuation or prevention of one or more symptoms associated with an RSV infection in a mammal.

An effective amount may be a maximum of 100 mg of antibody10 g / kg body weight, such as a maximum of 90, a maximum of 80, a maximum of 70, a maximum of 60, a maximum of 50, a maximum of 40, a maximum of 30, a maximum of 30 20, maximum 10, maximum 9, maximum 8, maximum 7, maximum 6, maximum 5, maximum 4, maximum 3, maximum 2, maximum 1, maximum 0.9, maximum 0.8, maximum 0.7, maximum 0.6, maximum 15, 0.5, maximum 0.4, 0.3, maximum 0.2, and maximum 0.1. mg per kg body weight.

In another embodiment, the effective amount is at least 0.01 mg of antibody per kg body weight, such as at least 0.05, 0.1, 0.2, 0.3, 0.4, 0, 5, 0.6, 0.7, 0.8.

Preferably, the effective amount is between 0.1 to 50 mg of

antibody per kg body weight. More preferably, the effective amount is between 1 and 20 mg of antibody per kg body weight.

The antibody may be administered at least once a year, such as 1,2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 times a year.

In particular, the antibody may be administered at regular intervals during the time of year when there is a high risk of contracting an RSV infection. Regular intervals can be weekly, biweekly, monthly, or bi-monthly.

Preferably, the mammal in the above embodiments is a human being.

human, a domestic animal or a pet.

In another embodiment, the mammal, patient of the method for preventing, treating or alleviating one or more symptoms associated with RSV infection, preferably has body weight above 40 kg.

In modalities in which the patient is a human being, he is preferably a premature baby, a child with chronic lung disease 5 or congenital heart disease. In alternative embodiments, the human is an immunocompromised adult, in particular a bone marrow transplant recipient, an elderly individual or an individual with chronic lung disease.

Diagnostic use

Another embodiment of the invention is directed to kit for

agnostic. Kits according to the present invention comprise an anti-RSV rpAB prepared according to the invention whose protein may be labeled with a detectable marker. or unlabeled for unlabeled detection for unlabeled detection. The kit can be used to identify individuals infected with RSV.

A diagnostic kit generally comprises the following: a) a capture antibody, b) a detector antibody, c) a positive control, and d) a negative control. Depending on the detection method, a single reagent may be used as either a capture antibody as a detector. A surface plasmon resonance (SPR) based method is an example in which a single antibody will be sufficient for specific antigen capture and detection of interest. The anti-RSV rpAb of the invention can be used as both capture antibody and detector antibody. As a capture antibody, unlabeled anti-RSV rpAb is adsorbed onto a solid support, for example, to surfaces of ELISA plate wells or microcounts for subsequent capture of RSV particles or antigens in patient samples. The captured antigen is then detected using a different antibody (detector) which is directly labeled or detected using a secondary conjugated antibody or reagent 30 of sufficient specificity. As a detector antibody, the anti-RSV rpAb of the invention may be used labeled or unlabeled and the amount of bound antibody detected directly (labeled rpAb) or using a secondary antibody or reagent (unlabeled rpAb).

The reading of the diagnostic kit may be based, for example, on the fluorescence detection of an attached fluorogenic marker or the absorbance of an added chromogenic substrate catalyzed by an enzyme conjugate to the detector antibody or secondary antibody (enzyme immunoassay). In an SPR1-based method the amount of bound antigen is detected in real time by changes in local refractive index as it binds to the adsorbed capture antibody. Regardless of the reading, by comparing signal strength to a standard curve 10 generated using the positive control, the amount of antigen present in the same sample can be determined.

Antibody molecules of the present invention and aspects related thereto

It should be noted that it is believed herein that the novel antibody molecules described herein contribute to the state of the art in its own right. Thus, the present invention also relates to any of the antibody molecules described herein as well as fragments and analogs of these antibodies, wherein said fragments or analogs at least incorporate the CDRs of the antibodies described herein.

For example, it has been found by the present inventors that al

Some of the entirely human antibody molecules that have been isolated from human donors include binding sites that exhibit extremely high improved kinetic profiles relative to the known prior art monoclonal antibodies with respect to antigen binding. Thus, while much focus is placed on polyclonal antibody compositions in the present disclosure, all the subject matter of polyclonal antibody use set forth herein is also relevant to any of the simple antibody molecules described herein - that is, all exposures that are related The formulation, dosage, administration, etc., which relate to the polyclonal antibody compositions of the present invention apply mutatis mutandis to the individual antibody molecules, antibody fragments and antibody analogs described herein, preferably also to the framework sequences.

Thus, the present invention also relates to an isolated human anti-RSV antibody molecule selected from the antibody molecules shown in Table 6 below, or a specifically binding fragment of said antibody molecule or a synthetic or semi-synthetic antibody analog. wherein said binding fragment or analog comprises at least the complementarity determining regions (CDRs) of said isolated antibody molecule. Often, framework regions from native human antibody variable regions will also be included in fragments or analogs, as antibody antigen specificity is known to be independent of the three-dimensional organization of CDRs and framework regions.

The term "isolated antibody molecule" means a collection of antibodies that are isolated from natural contaminants, and which exhibit the same amino acid sequence (that is, identical variable and constant regions).

Typically, any antibody molecule, fragment or analog is derived from the antibodies listed in Table 6, or includes the heavy chain CDR amino acid sequences included in one of SEQ ID 20 NOs; 1-44, and the accompanying light chain CDR amino acid sequences having a SEQ ID NO that is greater than the selected amino acid sequence of SEQ ID NOs: 1-44. This means that the antibody molecule, fragment or analog will include the cognate pairs of variable regions found in them of the 44 clones discussed above.

As mentioned above, various antibody molecules and

They exhibit very high affinities, so that the invention also pertains to an isolated antibody molecule, an antibody fragment, or a synthetic or semi-synthetic antibody analog, wherein the Fab having a dissociation constant Kd, for RSV G protein of maximum 500 30 nM when measured by plasmid resonance analysis on a Biacore 3000 using recombinant RESV G protein immobilized on the very low-density sento surface to avoid limitations on mass transport. The isolated antibody molecule, antibody fragment, or synthetic or semi-synthetic antibody typically exhibits a lower K0 of at most 400 nM, such as at most 300 nM, at most 200 nM, at most 100 nM, at most 1 nM at maximum 900 pM, maximum 800 pM, 5 maximum 700, pM maximum 600 pM, maximum 500 pM, maximum 400 pM, maximum 300 pM, maximum 200 pM, maximum 100 pM, maximum 90 pM , and at most 80 pM. Details concerning Biocore measurements are given in the examples.

Another embodiment of the invention relates to an isolated antibody molecule, an antibody fragment or a synthetic or semi-synthetic antibody comprising an antigen binding site identical to the antigen binding site in a Fab derived from a human antibody. wherein said Fab has a dissociation constant, KD, for RSV protein F of up to 500 nM when measured by performing surface plasmon resonance analysis on a Biacore 3000, a protein F of Recombinant RSV immobilized on sensor surface at very low density to avoid mass transport limitations. Such isolated antibody, antibody fragment or synthetic or semi-synthetic antibody typically exhibits a K0 of at most 400 nM, such as at most 300 nM, at most 200 nM, at most 100 nM, at most 1 nM, at most 900 pM, at 800 pM maximum 700 pM maximum 600 pM maximum 500 pM maximum 400 pM maximum 300 pM maximum 200 pM maximum 100 pM maximum 90 pM maximum 80 pM maximum 70 pM, maximum 60 pM, maximum 50 pM, maximum 40 pM, maximum 30 pM, maximum 25 pM, maximum 20 pM, maximum 15 pM, maximum 10 pM, maximum 9 pM, maximum 8 pM, maximum 7 pM, maximum 6 pM, and maximum 5 pM.

An especially useful antibody molecule or specifically binding fragment or synthetic or semi-synthetic antibody analog comprises the CDRs of a human antibody produced in clone No. 810, 818, 819, 824, 825, 827, 858 or 894.

As mentioned above, such useful antibody molecules of the present invention may be formulated in the same manner and for the same applications as the monoclonal formulations of the present invention. Thus, the present invention relates to an antibody composition comprising an antibody molecule, specifically binding fragment or synthetic or semi-synthetic antibody analog discussed in this section in admixture with a pharmaceutically acceptable carrier, excipient, vehicle or diluent. The composition may comprise more than one binding specificity, and may include, for example, 2 distinct antibody molecules of the invention and / or specifically binding fragments and / or analogs of synthetic or semi-synthetic antibodies of the invention. The composition may further comprise at least 3 distinct antibody molecules and / or specifically binding fragments and / or synthetic or semi-synthetic antibody analogs of the invention, and may therefore constitute a composition comprising at least 4, 5, 6, 7 , 9, 10, 11, 12, 13, 15, 16, 15 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 distinct antibody molecules and / or fragments and / or analogs of synthetic or semi-synthetic antibodies.

Especially interesting compositions include at least one RSV F protein binding antibody, fragment or analog of the invention and at least one RSV protein F binding antibody, fragment or analog of the invention.

Also a part of the invention is an isolated nucleic acid fragment encoding the amino acid sequence of at least one defined CDR of an antibody molecule of the present invention, such as a nucleic acid fragment encoding at least the CDRs of a antibody produced by one of the clones listed in Table 6. The nucleic acid fragment is typically DNA, but may also be RNA.

Another embodiment is an isolated nucleic acid fragment encoding CDR sequences of a heavy chain amino acid sequence set forth in any of DES ID NOs: 1-44, or an isolated nucleic acid fragment encoding CDR sequences. CDRs of a light chain amino acid sequence set forth in any of SEQ ID NOs: 89-132. Preferred nucleic acid fragments of the invention encode the CDR sequences of a heavy chain amino acid sequence set forth in any of SEQ ID NOs: 1-44 and set forth in amino acid sequences having a SEQ 5 ID NO that is greater than 88 the amino acid sequence selected from SEQ ID NOs: 1-44. This means, of course, that the nucleic acid fragment will encode the cognate pairs of variable regions found in them of the 44 clones discussed above. The nucleic acid fragment may therefore include coding sequences comprised in SEQ ID 10 NOs: 45-88 and / or 133-176.

Conveniently, nucleic acid fragments are introduced into a vector, which is also part of the present invention. Such a vector may be capable of autonomous replication, and is typically from the group consisting of a plasmid, a phage, a cosmid, a minichromosome, and a virus.

In the case where the vector is an expression vector, it will preferably have the following profile (see also an exemplary vector in Figure 3).

- in the 5 '-> 3' direction and operably linked at least one promoter to trigger expression of a first nucleic acid fragment discussed

above, which encodes at least one light chain CDR together with any required framework regions, optionally a nucleic acid sequence encoding a leader peptide, said first nucleic acid fragment, optionally a nucleic acid sequence encoding constant regions of an antibody, and optionally a nucleic acid sequence encoding a first terminator, and / or

- in the 5 '-> 3' direction and operably linked to at least one promoter for triggering expression of a second nucleic acid fragment of the invention encoding at least one heavy chain CDR together with any necessary framework regions, optionally one. sequence

nucleic acid encoding a leader peptide, said second nucleic acid fragment, optionally a nucleic acid sequence encoding constant regions, and optionally a nucleic acid sequence encoding a second terminator. Such a vector is especially useful if it can be used to stably transform a host cell, which can subsequently be cultured to obtain the recombinant expression product. Thus, the preferred vector is one that when introduced into a host cell is integrated into the host cell genome.

Thus, the invention also pertains to a transformed cell carrying the vector of the invention discussed in this section and also to a stable cell line carrying the vector and expressing the nucleic acid fragment of the invention discussed in this section. Both the transformed cell and the cell line optionally secrete or carry their recombinant expression product (i.e., the inventive antibody molecule, antibody fragment or analog) on their surface. 824 mAb and analogs

The present invention relates to monoclonal antibody 824 (mAb 824) and analogs thereof. The antibody light chain 824 has the amino acid sequence set forth in SEQ ID NO: 107. The heavy chain variable region has the amino acid sequence set out in SEQ ID NO: 19.

The invention relates to antibodies capable of competing with binding of antibody 824 as defined herein or with its Fab fragment. The antibody encoded by clone 824 as defined herein does not bind to particularly high affinity or potent recombinant RSV antigen. On the other hand, in virus neutralization assays (see Table 8), antibody 824 has a particularly low EC50 value against several different RSV isolates. When tested in an in vivo RSV infection model (mouse challenge model, Example 1, k-1), mAb824 showed a significantly greater reduction in virus burden than Synagis (Table 11b).

In providing the 824 antibody, the inventors have identified an epitope, which results in more efficient in vitro and in vivo neutralization than seen before for any single RSV epitope. By providing antibody 824, the inventors also made it possible to identify other antibodies that bind to the same epitope. Such other epitopes may be of any origin and include binding fragments as well as affinity matured antibodies. Antibodies capable of competing with antibody 5,824 can be identified in a cell competition assay (determination of relative epitope specificities) as described in Example 1, section g-4.

A preferred antibody capable of competing with antibody 824 is an anti-RSV antibody comprising a CDRH3 having the formula: CAX1X2X3X4X5X6PX7X8X9X10X11W,

where Xi to Xn are individually selected from the amino acid groups listed below,

Xi = R or K (ie positively charged at physiological pH);

X 2 = D, E, N or Q (ie reasonably bulky, polar amino acids);

X 3 = S, T, G or A (i.e. small, preferably polar amino acids);

X 4 = S, T, G or A (i.e. small, preferably polar amino acids);

X5 = N, Q, D or E (ie reasonably bulky, polar amino acids);

X6 = W, Y, F or H (i.e. bulky, aromatic amino acids);

X7 = A, G, V, or S (ie small amino acids);

X8 = G, A, V, or S (ie small amino acids);

Xg = Y, F, W or H (ie bulky, aromatic amino acids);

X-io = E or D (ie negatively charged at physiological pH); and

X11 = D, E, N or Q (ie reasonably bulky, polar amino acids);

and a CDRL3 described by the following formula: CX-1X2X3X4X5X6PX7TF where X1 to X11 are individually selected from the amino acid groups listed below:

X1 = Q or H (ie bulky, polar amino acids); X 2 = Q 1 E or H (i.e. bulky, polar amino acids);

X 3 = F, Y, W or H (ie bulky, aromatic amino acids);

X4 = N, Q or H (ie reasonably large, polar amino acids);

X5 = T, S, G or A (i.e. small, preferably polar amino acids);

Χδ = Y, F, W or H (ie bulky, aromatic amino acids); and

X7 = F, Y, W or H (ie bulky, aromatic amino acids).

Antibody binding specificity is mainly determined by the heavy and light chain CDR3 region. As is known in the art, certain substitutions may be made in an amino acid sequence without altering the three-dimensional structure of the protein. It is thus expected that mutations as described above can be made in the 824 antibody CDR3 sequences as defined herein at the same time the 824 antibody specificity and potency are maintained.

Introduction of amino acid changes in a protein is known in the art. Antibodies with altered CDR3s compared to antibody 824 can be made and they can be tested in virus neutralization assays as described in the examples. Conservation and binding specificity can be verified in a competition assay 20 with antibody 824.

Preferably, the anti-RSV antibody comprises the CDR1 and CDR2 regions of the Vh and V1 pair as set forth in SEQ ID NOs: 232, 317, 487, and 572, and a CDRH3 region having the formula CaR1D2S3S4N5W6Pa7G8Y9E1ODiiW (SEQ ID NO: 402), and a CDRL3 region having the formula formula CQ1Q2F3N4T5Y6PF7TF (SEQ ID NO: 657).

In preferred embodiments, CDRH3 has the amino acid sequence set forth in SEQ ID NO: 402 and / or CDRL3 has the amino acid sequence set out in SEQ ID NO: 657.

In one embodiment, the antibody comprises the VH region (SEQ ID NO: 19) of antibody 824. The monoclonal antibody may also comprise the VL region (amino acids 1 to 107 of SEQ ID NO: 107) of antibody 824. Preferably, the The antibody comprises the light chain (SEQ ID NO: 107) of antibody 824.

The antibody may comprise CH as defined in SEQ ID NO: 178. Other heavy chain constant regions may be used.

Preferably, the monoclonal antibody is capable of neutralizing RSV A and B subtypes in a virus neutralization assay. The neutralizing potency of the monoclonal antibody is preferably comparable to the potency of mAB925.

Preferably, the antibody is capable of providing a reduced

RSV virus burden in the lungs of an RSV-infected mammal. Preferably, this reduction is significant compared to the reduction provided by Synagis, and more preferably, the reduction is comparable to the reduction provided by mAb824.

MAb824 antibody and sequence-based antibodies

mAb824 CDRs may be combined with and one or more additional anti-RSV antibodies to provide a polyclonal antibody. Such polyclonal antibody or antibody mixture (s) may be less susceptible to escape mutants.

One or more additional anti-RSV antibodies may be selected.

of the group consisting of human antibodies, humanized antibodies and chimeric human / chamoid antibodies. For example, the additional antibody may be Palivizumab (Synagis) or MEDI-524 (Numax).

Preferably, the one or more additional antibodies are selected from the group consisting of the antibody molecules shown herein in Table 6, or a specifically binding fragment of said antibody molecule or a synthetic or semi-synthetic antibody analog, wherein said fragment The binding or analogue moiety comprises at least the complementarity determining regions (CDRs) of said isolated antibody molecule 30 except that an antibody has the CDRs of clone 824.

Also provided are nucleic acids comprising an amino acid sequence of at least one CDR defined in this section.

The nucleic acid fragment may encode the CDR sequences of a heavy chain amino acid sequence set forth in SEQ ID NO: 19. The isolated nucleic acid fragment may encode the CDR sequences of the light chain amino acid sequence set forth in SEQ ID. NO: 107.

Preferably, the isolated nucleic acid fragment encodes the heavy chain amino acid sequence CDR sequences set forth in SEQ ID NO: 19 and the accompanying heavy chain CDR 10 amino acid sequences. In another preferred embodiment, the nucleic acid fragment includes coding sequences comprised in SEQ ID NO: 63 and / or 151.

Isolated nucleic acids and fragments can be inserted into a vector.

The vector may be capable of autonomous replication and may be if

taught from the group consisting of plasmid, phage, cosmid, a minichromosome, and virus.

In another mode the vector comprises:

- in the 5 '- »3' direction and operably linked to at least one promoter to trigger expression of a first nucleic acid fragment as

described in this section, which encodes at least one light chain CDR derived from clone 824 together with the necessary framework regions, optionally a nucleic acid sequence encoding a leader peptide, wherein said first nucleic acid fragment, optionally 25 a. nucleic acid sequence encoding constant regions, and optionally a nucleic acid sequence encoding a first terminator, and / or

- in the 5 '- »3' direction and operably linked to at least one promoter to trigger expression of a second nucleic acid as described herein.

section, which encodes at least one clone 824-derived heavy chain CDR together with the necessary framework regions, optionally a nucleic acid sequence encoding a leader peptide, wherein the second nucleic acid fragment additions, optionally an acid sequence nucleic acids encoding constant regions, and optionally a nucleic acid sequence encoding a second terminator.

The vector when introduced into a host cell can

be integrated into the host cell genome.

Also provided is a transformed cell carrying the vector as described in this section, and a stable cell line carrying the vector as described in this section and expressing a nucleic acid fragment as described in this section, and optionally secreting or transporting its vector. recombinant expression product on its surface.

EXAMPLE 1

This example is a collection of methods applied to illustrate the invention.

The. Blood Lambda negative blast classification

from the donor

Peripheral blood mononuclear cells (PBMC) were isolated from donor blood using Lymphoprep (Axis Shield) and gradient centrifugation according to the manufacturer's instructions. PBMCs alone were either cryopreserved in FCS: 10% DMSO at -150 ° C or used directly. The B cell fraction was labeled with anti-CD19 antibody and isolated from the PBMC fraction using magnetic cell classification (MACS). PBMCs (1x10 6 cells) were incubated with anti-CD19-FITC conjugated antibody (BD Pharmingen) for 20 minutes at 4 ° C. Cells were washed twice in and resuspended in MACS buffer (Miltenyl Biotec). Anti-FITC Microcounts (Miltenyl Biotec) were mixed with labeled cells and incubated for 15 minutes at 4 ° C. The washing procedure was repeated before cell / bead suspension was applied to an LS MACS (Miltenyl Biotec) column. The CD19 positive cell fraction was eluted from the column according to the manufacturer's instructions and either stored in FCS-10% DMSO or sorted directly into single cell.

Plasma blasts were selected from the CD19 + B cell fraction by fluorescence activated cell classification (FACS) based on the CD19, CD38 and CD45 cell surface protein expression profile. CD19 is a B cell marker that is also expressed in plasma cell precursors, while CD38 is highly expressed in plasma blasts and plasma cells. Plasma blasts apparently have a somewhat lower CD19 and CD45 expression than the rest of the CD19 + cells, which allows the separation of a discrete population. Cells were washed in FACS buffer (PBS; 1% BSA) and stained for 20 minutes with anti-CD19-FITC, anti-CD38-APC, anti-Lambda-PE (BD Pharmingen). Lambda light chain staining has been included to allow the exclusion of cells that cannot serve as a template for PCR (see Section c). The colored cells were washed and resuspended in FACS buffer.

Cell flow during FACS was adjusted to approximately 200 events / second and cell concentration was 5x105 / mL to achieve high plasma cell rescue. The following set of ports was used. Each door is a daughter of the previous one.

Port 1: FSC / SCC port. The lymphocyte population having the highest FSC was selected, thus ensuring the classification of living cells.

Port 2: SSChSSCw. This port ensured single cell classification (double discrimination);

Port 3: Events representing the plasma blasts were placed at the port in the dotted CD38 / CD19 plot as intermediate CD38 High / CD19.

Port 4: Since the PCR procedure described in Section c amplifies only Capa light chains, Lambda negative events were placed on the door in a dotted Lambda / CD19 plot.

Alternatively or on port 3, plasma blasts

can also be identified as high CD38 and intermediate CD45 in a dotted CD45 / CD38 plot. This will require staining of the cells with anti-CD45-PerCP.

The resulting population that met these four criteria was sorted from single cells into 96-well PCR plates containing the sorting buffer (see Section c). The plates containing the cells were stored at -80 ° C.

B. ELISpot

ELISpot was used to estimate the percentage of anti-RSV antibody-expressing plasma blasts in the cell samples obtained, ie PBMC, MACS purified CD19 + cells, or a population of FACS-classified plasma blasts. Nitrocellulose 96-well plates (MiIIipore) were coated with a 25 μg / mL solution of inactivated RSV Long particles (HyTest). The wells were blocked by incubation for 1 hour at 37 ° C. Plates were washed and cell samples were added in RPMI culture medium to each well, followed by incubation under standard tissue culture conditions for 24 hours. Secreted RSV-specific antibodies will bind to virus-immobilized particles surrounding the antibody-producing cell. Cells were removed by washing three times in PBS;

0.01% Twee20 and three times in PBS. HRP-conjugated anti-human IgG (H + L) (CaITag) and HRP-conjugated anti-human IgA (Serotec) were added and allowed to react with the immobilized antibodies for 1 hour at 37 ° C. The washing procedure was repeated and the N, N-DMF-solubilized chromogen (3-amino-9-ethylcarbazole) substrate was added. Color development was terminated after 4 minutes by addition of H2O. Red dots were identified at sites where antigen-specific antibody-secreting cells had been located.

ç. Binding of cognate pairs of Vh and Vl

The binding of Vh and Vl coding sequences was performed on the simple cells obtained as described in Section a, facilitating the cognate pairing of Vh and Vl-coding sequences. The procedure used a two-step PCR procedure based on RT- One-step multiplex overlap-extension PCR followed by Nested PCR. Primer mixtures used in the present example amplify only Capa light chains. Primers capable of amplifying Lambda light chains could, however, be added to the 5 multiplex primer mix and the Nested PCR primer mix, if desired. If Lambda primers are added, the classification procedure in Section a must be adapted so that Lambda positive cells are not deleted. The principle for binding cognate Vh and Vl sequences is illustrated in Figure 2.

The 96-well PCR plates produced in Section a were

thawed cells and sorted cells served as a template for multiplex overlap-extension RTPCR. The sort buffer added to each well prior to single cell sorting contained reaction buffer (OneStep RT-PCR Buffer; Qiagen), RT-PCR primers (see Table 2) and RNase inhibitor (RNasin, Promega). This was supplemented with OneStep RT-PCR Enzyme Mix (25 x dilution; Qiagen) and dNTP mix (200 μΜ each) to obtain the given final concentration in a 25 μί reaction volume.

Plates were incubated for 30 minutes at 55 ° C to allow reverse transcription of RNA from each cell. Following RT, the plates were subjected to the following PCR cycle: 10 minutes at 94 ° C, 35x (40 seconds at 94 ° C, 40 seconds at 60 ° C, 5 minutes at 72 ° C), 10 minutes at 72 ° Ç.

PCR reactions were performed on the H20BIT Thermal cycler with a Peel Seal Basket for 24 96-well plates (ABgene) to facilitate high throughput. The plates were stored at -20 ° C after cycling.

Table 2: Mixture of multiplex overlap extension RT-PCT primers

Cone name. Start SEQ Sequence nM ID NO: creator VH Set Name Cone. Inhibit SEQ Sequence nM ID NO: CH-IgG Creator 0.2 GACSGATGGGCCCTTGGTGG 179 CH-IgA 0.2 GAGTGGCTCCTGGGGGAAGA 180 VH-1 0.04 T ATT CCCATGGCGCGCCCAGRT G-181 CAGCTGGTGCTG CCTG CCTG CCTGT CCTG CCTG CCTG CCTGCGT - 182 TRCAGT VH-3 0.04 T ATT CCCATGGCGCGCCCAGRT CACCTT GA-183 CAGGAGT VH-7 0.04 TATTCCCATGGCGCGCCGARGTG-187 CAGCTGGTGCAGT VH-7 0.04 TATTCCCATGGCGCGCCCAGGTACAGCTG- 188 CAGCAGTC LC set CK1 0.2 AT AT AT AT GGGCCGCTT GGT TGA GGTTAT GGTT GGTT GGTGTT G ACCCAGT CT VL-2 0.04 GGCGCGCCATGGGAATAGCTAGCCGATGTTGT 191 GAT GACT CAGT CT VL-3 0 04 GGCGCGCCATGGGAATAGCTAGCCGAAATTGT 192 GWT VL-L ACRCAGT CT 4 0.04 193 GAT GGCGCGCCATGGGAATAGCTAGCCGATATTGT GACCCACACT VL-194 5 0.04 GGCGCGCCATGGGAATAGCTAGCCGAAACGA- CACT CACGCAGT VL-6 0.04 195 L GGCGCGCCATGGGAATAGCTAGCCGAAATTGT CT CT GACT CAGT W = A / T, R = A / G, S = G / C

For the nested PCR step, 96-well PCR plates were prepared with the following mixture in each well (20 μΙ_ reactions) to obtain the given final concentration: 1x FasStart Buffer (Roche), dNTP Mix (20 μΜ each) , nested primer mix (see Table 3), DNA Polymerase Phusion (0.08 U; Finnzymes) and FastStart High Fidelity Enzyme Combination (0.8 U; Roche). As a template for Nested PCR, 1 μΙ_ is transferred from multiplex overlap-extension PCR reactions. Nested PCR plates were submitted to the following cycle; 35x (30 seconds at 95 ° C, 30 seconds at 60 ° C, 90 seconds at 72 ° C) for 10 minutes at 72 ° C.

Randomly selected reactions were analyzed on 1% agarose gel to verify the presence of an approximately 1070 bp overlap-extension fragment.

Plates were stored at -20 ° C until further processing.

PCR fragments.

Table 3: Nested Primer Set Cone Name. Final. Primer Sequence SEQ ID CH2 0.2 nM 196 ACCGCCTCCACCGGCGGCCGCTTATTAAC GAAGCT ACT CTT GTT CT JSTCC PJ 1-2 0.2 L GGAGGCGCTCGAGACGGTGAC- 197 CAG GTG CC 3 PJ PJ CATTGTCCC 0.2 GGAGGCGCTCGAGACGGTGAC- 198 4-5 0.2 199 GGAGGCGCTCGAGACGGTGAC- CAGGGTTCC PJ 6 0.2 GGAGGCGCTCGAGACGGT-200 .... GACCGT GGT CCC d. Inserting coding pairs of Vh and Vl into a vector of

selection

In order to identify antibodies with binding specificity

For RSV particles or antigens, the Vh and Vl coding sequences obtained as described in Section c were expressed as full-length antibodies. This involved insertion of the Vh and Vl coding pairs of the repertoire into an expression vector and transformation into a host cell. A two-step cloning procedure was employed to generate a repertoire of expression vectors containing the linked Vh and Vl coding pairs. Statistically, if the expression vector repertoire contains ten times as many plasmids that the number of paired Vh and Vl PCR products used to generate the selection repertoire, there is a 99% probability that all single gene pairs are represented. . Thus, if 400 overlap-extension V gene fragments were obtained in Section c, a repertoire of at least 4,000 clones was generated for selection.

In summary, the repertoires of Vh and Vl encoding pairs

Nested PCR ligands in Section c were pooled (without mixing the different donor pairs). PCT fragments were cleaved with Xho \ and Not \ DNA endonucleases at recognition sites introduced at the PCR product termini. Cleaved and purified fragments were ligated into a Xho \ INot \ digested mammalian IgG expression vector (Figure 30 by standard ligation procedures. The ligation mixture was electroporated into E. coli and added to 2x YT plates containing the appropriate antibiotic and incubated at 37 ° C overnight The amplified vector repertoire was purified from cells recovered from the plates using 20 standard DNA purification methods (Qiagen) Plasmids were prepared for insertion of promoter fragments. by cleavage leader using / Ascl and NheI endonucleases.The restriction sites for these enzymes were located between the Vh and VL coding gene pairs.After vector purification, a di-managed bidirectional mammalian leader promoter fragment of Asc? NheI / Ascl and NheI restriction sites were inserted by standard ligation procedures.The ligated vector was amplified in E. coli and the plasmid was purified by mRNA. The generated selection vector repertoire was transformed into E. coli by conventional procedures. The colonies obtained were consolidated in 384-well and 30-stored master plates. The number of arranged colonies exceeded the number of PCR products at entry at least 3-fold, thus giving a 95% probability for the presence of all unique V gene pairs obtained in section C.

and. Selection

Bacterial colonies arranged in Section d were inoculated into 384-well similar plate culture medium and grown overnight. DNA for transfection was prepared from each well in the cell culture plate. The day before transfection 384-well plates were seeded with CHO-FIp-In cells (Invitrogen) in 3,000 cells / well in 20 μΙ culture medium. Cells were transfected with DNA using Fugene6 (Roche) according to the manufacturer's instructions.

After 2-3 days incubation, supernatants containing life-size antibodies were collected and stored for selection purposes.

Selection was performed using the Applied Biosystems 8200 FMAT® System, a FLISA homogeneous bead-based soluble capture assay (with fluorescent bound immunosorbent) (Swartzaman et al. 15 1999, Anal Biochem. 271: 143-151). Several antigens, including virus particles, recombinant G protein and biotinylated peptides derived from RSV antigens, were used for selection. The peptides were derived from the conserved region (amino acids 164-176) and the cysteine nucleus region (amino acids 171-187, Long strain and 18537) of the G protein and the extra20 cellular region of the SH protein (amino acids 42-64 of the A2 and 42 strain -65 of strain 18537). Inactivated RSV Long strain (HyTest) virus particles were immobilized on polystyrene beads by incubating 300 μί of 5% w / v beads (6.79 μητι in diameter, Spherotech Inc.) with 300 μί of virus stock (protein concentration: 200 μg / mL). Soluble recombinant G protein (amino acids 66-692 of strain 18537 sequence) was similarly immobilized directly on polystyrene beads, while biotinylated peptides were captured on streptavidin-coated polystyrene beads (diameter 6.0-8 , 0 μιη, Gerlinde Kisker) at saturate concentrations. The coating mixture was incubated overnight and washed twice in PBS. The beads were resuspended in 50 ml PBS containing 1% bovine serum albumin (PBS / BSA) and 5 µl of Alexa 647 goat anti-human IgG pool (Molecular probes). Ten microliters of resuspended coating mixture was added to 20 µl antibody-containing supernatant in FMAT-compatible 384-well plates and incubated for approximately 12 h, after which fluorescence on the bead surface in individual wells was measured. A fluorescence event was recognized as positive if its intensity showed at least six deviation patterns above the residual baseline.

The resulting clones in the main ranges were taken from the original master plates and collected on new plates. DNA was isolated from these clones and subjected to DNA sequencing of the V genes. The sequences were aligned and all single clones were selected.

The selected clones were further validated. In summary, 2x10 6 293 Freestyle cells (Invitroen) were transfected with 1.7 μg of DNA from the selected clones and 0.3 μg of plasmid pAdVAntage (Promega) in 2 mL of Freestyle medium (Invitrogen) according to 15 instructions from manufacturer. After two days, the supernatants were tested for IgG expression and reactivity with the different antigens used for primary selection as well as recombinant purified protein F and a produced protein G E. coli fragment (amino acids 127-203 of the strain 18537) by FLISA and / or ELISA. Antibody supernatants were tested in serial dilutions allowing clones to be classified according to antigen reactivity.

f. Clone Repair

When using a multiplex PCR approach as described in Section c, some degree of cross-sensitization of the family of 25 intra- and intergenes V is expected due to the high degree of homology. Cross-sensitization introduces non-naturally occurring amino acids into the immunoglobulin structure with several potential consequences, for example structural changes and increased immunogenicity, all resulting in decreased therapeutic activity.

In order to eliminate these disadvantages and to ensure that

selected clones mirror the natural humoral immunoresponse, such cross-sensitization mutations were corrected in a process called clone repair.

In the first step of the clone repair procedure, the Vh sequence was PCR amplified with a set of primers containing the sequence corresponding to the Vh gene of interest originated therein, 5 thereby correcting any mutations introduced by cross-sensitization. The PCR fragment was digested with Xho / e / Asci and ligated back to the Xho / Asc / digested mammalian expression vector (Figure 3) using standard ligation procedures. The ligated vector was amplified in E. coli and the plasmid was purified by standard methods. The ligated vector was amplified in E. coli and the plasmid was purified by standard methods. The Vh sequence was sequenced to verify correction and the vector was digested with Nhe \ INot \ to prepare it for light chain insertion.

In the second step, the complete light chain was PCR amplified with a set of primers containing the sequence corresponding to the V1 gene of the clone of interest originated therein,

thereby correcting any mutations introduced by cross-sensitization. The PCR fragment was digested with Nhe \ INot \ and ligated to the Vh containing vector prepared above. The ligation product was amplified in E. coli and the plasmid was purified by standard methods. Subsequently, the light chain was sequenced to verify correction.

In the event that the Cape constant region of a selected clone contained mutations introduced during gene amplification as described in Section c, it was replaced by an unchanged constant region. This was done in overlapping PCR in which the repaired Vl 25 gene (amplified without the constant region) was fused to a constant region with the correct sequence (obtained in a separate PCR). The entire sequence was amplified and cloned into the Vh-containing vector as described above and the repaired light chain was sequenced to verify correction.

g. Generation of a polyclonal cell line

Generation of a polyclonal expression cell line producing a recombinant polyclonal antibody is a multi-step procedure involving the generation of individual expression cell lines in which each expresses a unique antibody from a Vh and VL gene sequence. . Simple. The polyclonal cell line is obtained by mixing individual cell lines and distributing the mixture in ampoules, thereby generating a polyclonal research cell bank (pRCB) or a master cell bank (pMCB) from which a Polyclonal work cells (pWCB) can be generated by expanding search or master cell bank cells. Generally, polyclonal cells from pRCB are used directly 10 without generating a pWCB.

The individual steps in the process of generating a polyclonal cell line are described below.

g-1 Transfection and selection of mammalian cell lines The Flp-In CHO (Invitrogen) cell line was used as the starting cell line. In order to obtain a more homogeneous cell line, the Flp-In CHO cell line was subcloned by limited dilution and several clones were selected and expanded. Based on the growth behavior, one clone, CHO-FIp-IN (019), was selected as the starting cell line. CHO-FiIp-In (019) cells were cultured as adherent cells in HAM-F12 with 10% fetal calf serum (FCS).

Individual plasmid preparations, each containing a pair of selected and repaired Vh and Vl coders obtained from Section f, were cotransfected with plasmid encoding Flp 25 recombinase into -19x106 CHO-FIp-In (019) cells (for For more details, see WO 04/061104) in a T175 flask using Fugene6 (nunc). Recombinant cell lines were selected by culture in the presence of 500 μg / mL Geneticin, which was added 48 hours after transfection. About two weeks later, the clones appeared. Clones were counted and cells were trypsinated and then cultured as clone assemblies expressing one of the RSV-specific antibodies.

g-2 Serum-free suspension culture adaptation Cell cultures expressing individual adherent anti-RSV antibody were trypsinated, centrifuged and transferred to separate 1.15x106 cell / mL shake flasks (250 mL) in free medium of appropriate serum (Excell302, JRH Biosciences; 500 pg / ml geneticin, 5 anti-caking agent (1: 250) and 4 mM L-glutamine. Cell growth and morphology were followed for several weeks. At 6 weeks, the cell lines usually showed good and stable growth behavior without doubling the times below 30 hours and the adapted individual cell lines were then cryopreserved in 10 multiple ampoules.

Individual antibodies expressed during adaptation were purified from supernatants using the method described in Section i. Purified antibody was used for characterization of antigen specificity and biochemical properties as described below.

g- 3 Characterization of cell lines

All individual cell lines were characterized with respect to antibody production and proliferation. This was performed with the following trials:

Production:

The production of recombinant antibodies from cell lines

Individual expression lines were followed during adaptation by Cover specific ELISA. ELISA plates were coated with purified goat anti-human Fc antibody (Serotec) in carbonate buffer, pH 9.6. Plates were washed 6 times with wash buffer (PBS; 25 0.05% Tween 20) and blocked by incubation for 1 h in wash buffer containing 2% skimmed milk. Supernatants from cell culture media were added and incubation extended for 1 hour. Plates were washed 6 times in wash buffer and secondary antibodies (Goat Anti-Human Goat Kappa, Serotec) were added and incubation repeated. After vigorous washing, the ELISA was developed with TMB substrate and the reaction was terminated by addition of H2SO4. The plates were read at 450 nm. In addition, intracellular staining was used to determine general expression level as well as to determine homogeneity of cell population relative to recombinant antibody expression. 5X105 cells were washed in ice cold FACS buffer (PBS: 2% FCS) prior to fixation by incubation in CeIIFix (BD-Biosciences) for 20 minutes. The cells were precipitated and permeabilized in ice cold methanol for 10 minutes and washed twice in FACS buffer. The suspension was a fluorescently labeled antibody (Bode F (ab ') 2 Fragment, IgG (H + L) anti-human PE, Beckman Coulter) was added. After 20 minutes on ice, the cells were washed and resuspended in FACS buffer followed by FACS analysis.

Proliferation:

Aliquots of cell suspensions were taken two to three times a week and cell number, cell size and viability were determined by Vi-Cell XR analysis (Beckman Coulter Cell Viability Analyzer). The doubling time for cell cultures was calculated using cell numbers derived from Vi-Cell measurements.

g-4 Antigen specificity characterization of antibodies

individual

The antigen and epitope specificity of individually expressed antibodies were evaluated to allow the generation of an anti-RSV rpAb with a well-characterized specificity. As described in Section e, antibodies identified during selection were validated by assessing their specificity for single RSV antigens (recombinant G protein, recombinant or purified protein F) or peptide fragments thereof (conserved region and cysteine nucleus motif). of protein G, subtype A and subtype B, linear epitopes on protein F, and the extracellular domain of protein SH, subtype AeB) by FLISA, ELISA 30 and surface plasmon resonance (SPR; Biacore). Epitope specificities were determined in ELISA by competition with well-characterized commercial antibodies, some of which are shown in Table 4. Not necessarily all antibodies shown in Table 4 were used in the characterization of each individual antibody of the present invention, and potentially others. Antibodies or antibody fragments which have been characterized with respect to the antigen, antigenic site and / or epitope to which they bind may also be used. In summary, antibodies or antibody fragments used to block epitopes were incubated with large excess immobilized antigen (RSV Long particles, HyTest), ie concentrations 100 times giving 75% of the maximum binding as determined empirically (Ditzel et al., J. Mol. Biol. 1997, 267: 684-695). After washing, individual antibody clones were incubated with the blocked antigen at various concentrations and any bound human IgG was detected using goat anti-human HRP conjugate (Serotec) according to standard ELISA protocols. Epitope specificities were further characterized by pair-type competition between different clones in Biacore using saturating (empirically determined) concentrations of both blocking and probe antibodies. Purified protein F or G immobilized by direct amine coupling (Biacore) was used as antigen. In both ELISA and Bioacore-based mappings, the reduced binding following epitope blocking is compared to non-competing binding.

Table 4: Epitope Mapping Monoclonal Antibodies

of anti-F and anti-G antibodies

MAb / Fab Antigen Site Antigen Epitope (aa) Ref. 131-2a F F1 F1a 1,2 9C5 F F1 F1a 5 92-11c F F1 F1b 1,2 102-1 Ob F F1 F1c 1,2 133-1h FC F2 1,2,3 130-8f FC F2 (241/421) 1,2,3,4 143-6c FA / ll F3 1,2,3 Palivizumab FA / I I (272) 8 1153 FA / ll (262) 3.4 MAb / Fab Antigen Site Antigen Epitope (aa) Ref. 1142 FA / ll 3 1200 FA / ll (272) 2.4 1214 FA / ll (276) 3.4 1237 FA / ll (276) 3.4 1129 FA / ll (275) 3.4 1121 FA / ll 3 1112 FB / l (389) 3.6 1269 FB / l (389) 3.6 1243 FC (241/421) 3.6 Fab 19 FA / ll (266) 7 RSVF2-5 F IV (429) 4 Mab19 F IV (429) 12 7,936 FV (432-447) 13 9,432 F Vl (436) 13 63-1 Of G (A) G11 GCRR (A171-187) 1.2 130-6d G (A) G12 (A174-214) 1.2 .9 131-2g G (A + B) G 13 (150-173) 1.2.9 143-5a G (A + B) G 5a 2 L9 G (A + B) Preserved (164-176) 14,15 8C5 G ND 5 1C2 G (A) ND GCRR (A172-188) 10,11 3F4 G (A) ND 10,11 4G4 G (A) ND GCRR (A172-188) 10,11 The "Antigen" column "indi RSV-associated antigen

Mab / Fab, and if a subtype specificity is known, this is indicated in (). The "Epitope (aa)" column indicates the name of the MAb / Fab recognized epitope, still at () resulting amino acid positions in RSV escape mutants, or protein peptides / fragments with respect to which binding has been shown. indicated. The numbered references (Ref.) Given in Table 4 correspond to:

1. Anderson et al., J. Clin. Microbiol. 1986, 23: 475-480. 2. Anderson et al., J. Virol. 1988, 62: 1232-4238.

3. Beeler & van Wyke Coelingh, J. Virol. 1989, 63: 2941-2950.

4. Crowe et al., IADB 1998, 177: 1073-1076.

5. Sominina et al., Vestn Ross Akad Med Nauk 1995, 9: 49-54.

6. Collins et al., Fields Virology, p. 1313-1351.

7. Crowe et al., Virology 1998, 252: 373-375.

8. Zhao & Sullender, J. Virol. 2004, 79: 3962-3968.

9. Sullender, Virology 1995, 209: 70-79.

10. Morgan et al., J. Gen. Virol. 1987, 68: 2781-2788.

11. McGIIII et al., J. Immunol. Methods 2005, 297: 143-152.

12. Arbiza et al., J. Gen. Virol. 1992, 73: 2225-2234.

13. Lopez et al J. Virol. 1998, 72: 6922-6928.

14. Walsh et al., J. Gen. Virol. 1989, 70: 2953-2961.

15. Walsh et al., J. Gen. Virol. 1998, 79: 479-487.

In addition, antibody clones were also characterized

in terms of binding to human laryngeal epithelial HEp-2 cells (ATCC CLL-23) infected with different RSV strains (Long, B1, or 18537) by FACS and / or ELISA. Binding to Mock-infected HEp-2 cells were analyzed similarly.

In summary, for the FACS assay, Hep-2 cells were infected with

RSV Long strain (ATCC Number VR-26) or RSV B1 strain (ATCC Number VR-1400) in serum-free medium at a rate of

0.1 pfu / cell per 24 (Long strain) or 48 hours (B1 strain). Following displacement and washing, cells were dispensed into 96-well plates and 25 incubated with dilutions (4 pM-200 μΜ) of individual anti-RSV antibodies for 1 hour at 37 ° C. Cells were fixed in 1% formaldehyde and antibody bound to the cell surface was detected by incubation with 1% anti-human IgG-PE conjugate (Beckman Coulter) for 30 minutes at 4 ° C.

For the ELISA assay, HEp-2 cells were infected with either RSV Long strain or strain 18537 (ATCC Number VR-1580) in serum free medium at a rate of 0.01 pfu / cell. After two hours of incubation, medium with 10% fetal calf serum was added and cells were incubated for an additional 45 hours (Long strain) or 70 hours (strain 18537). After washing, cells were incubated with dilutions of anti-RSV antibodies (0.1 μΜ - 0.03 nM) for 1 hour at room temperature. Cell surface bound antibody was detected by incubation with 5 goat F (ab) 2 anti-human IgG-HRP conjugate (Jackson ImmunoResearch) for 1 hour at room temperature, followed by addition of TMBplus (Ken-Em -Tec). After 10 minutes of incubation, the reaction was terminated with H2SO4 and the absorbance measured. Such cellular assays may also be used as a competition assay for determining relative epitope specificities as described for the virus particle ELISA and SPR assay described above.

Selected clones identified as specific for protein G were also tested for cross-reactivity with recombinant human fractalcin (CX3CL1) by ELISA. The anti-human CX3CL1 / Fractalcin monoclonal antibody (R&D systems) was used as positive control.

g- 5 Characterization of binding kinetics of individual antibodies

Kinetic analysis of the antibodies of the invention was performed using surface plasmon resonance analysis on a Biacore 3000 (Biacore AB, Uppsala, Sweden) using recombinant antigens immobilized on the very low density sensing surface to avoid limitations in mass transportation. Analysis was performed with Fab fragments prepared from individual antibody clones using the ImmunoPure Fab Preparation Kit (Pierce). In summary, a total of 200 recombinant protein F resonance units (RU) or a total of 50 recombinant protein G RU was conjugated to a CM5 chip surface using the Amine Coupling Kit (Biacore) according to the instructions of the manufacturer. Fab fragments were injected onto the chip surface in serial dilutions, starting from an optimized concentration that did not result in RUmax values above 25 when tested on the chip with immobilized protein. Association rate constant (ka) and dissociation constant (kd) were evaluated globally using the default 1: 1 association (Langmuir) and dissociation models in the BIAevaIuation 4.1 software (BlAcore).

By performing kinetics analysis on Fab1 fragments it is ensured that the data obtained truly reflects the binding affinities with respect to the RSV protein. If complete antibodies were used, the data would reflect binding avidity, which cannot readily be translated to a significant extent of the exact nature of antibody versus antigen binding characteristics.

g- 6 Characterization of antibody biochemical properties

individual

Heterogeneity is a common phenomenon in antibodies and recombinant proteins. Antibody modifications typically occur during expression, for example, post-translational modifications such as N15 glycosylation, proteolytic fragmentation, and N and C-terminal heterogeneity resulting in size or charge heterogeneity. In addition, modifications such as methionine oxidation and deamidation may occur during long and short term storage. Since these parameters need to be well-defined for therapeutic antibodies, they were analyzed prior to the generation of polyclonal cell line.

Methods used for the characterization of purified individual antibodies (see Section i) included SDS-PAGE (reducing and non-reducing conditions), weak cation exchange chromatography (IEX), size exclusion chromatography (SEC), and RP-HPLC. (reducing and not 25 reducing conditions). Analysis of SDS-PAGE under reducing and non-reducing conditions and SEC indicated that the purified antibodies were certainly intact with minute amounts of fragmented and aggregated forms. IEX profile analysis of the purified antibodies resulted in single peak profiles or multiple peak chromatograms, indicating charge heterogeneity in those particular antibodies. Antibody preparations resulting in multiple peaks in IEX analysis and / or aberrant migration of both light and heavy chains on SDS gels, or unusual RPHPLC profiles were analyzed in detail for N-termini by N-terminal sequencing and for heterogeneity caused by differences in oligosaccharide profiles. In addition, selected antibodies were screened for the presence of variable chain N-glycosylation sites using enzyme treatment and subsequent SDS-PAGE analysis.

g- 7 Establishment of a polyclonal cell line for production of recombinant anti-RSV polyclonal antibody

From the collection of established expression cell lines, a subset is selected to be mixed for generation of a polyclonal cell line and the polyclonal research / master cell bank (pRCB / pMCB). Selection parameters can be set according to the use of the polyclonal antibody to be produced from the polyclonal cell line and the execution of individual cell lines. Generally, the following parameters are considered:

· Characteristics of cell lineage; to optimize stability

Polyclonal cell line integrity, single cell lines with no doubling times between 21 and 30 hours and antibody productivity above 1 pg / cell / day are preferred.

• Reactivity; The antigens / antigenic sites against which anti-RSV rpAb will exert reactivity are carefully considered.

• protein chemistry; Preferably antibodies with well-defined biochemical characteristics are included in the final anti-RSV rpAb.

Selected individual cell lines each expressing a recombinant anti-RSV antibody are thawed and expanded at 37 ° C in serum free medium in shake flasks to reach at least 4x108 cells of each clone having a population doubling time of 21. at 34 hours. Viability is preferably in the range of 93% to 96%. The polyclonal cell line is prepared by mixing 2x10 6 cells from each cell line. Polyclonal cell line 30 is distributed in freezing ampoules containing 5.6x107 cells and cryopreserved. This collection of flasks with a polyclonal cell lineage is referred to as the research / master cell bank (pRCB / pMCB) from which the polyclonal working cell bank (pWCB) can be generated by expanding a pRCB ampoule. / pMCB to reach a sufficient number of cells to form a working cell bank (pWCB) of approximately 200 ampoules with the same cell density as pRCB / pMCB ampoules. Cell bank samples are tested for mycoplasma and sterility.

H. Expression of a recombinant polyclonal anti-RSV antibody

Batches of recombinant polyclonal anti-RSV antibodies are

produced in 5 liter bioreactors (B. Braun Biotech International, Melsungen, Germany). Briefly, the pRCB or pWCB flasks are thawed and expanded in shaker flasks (Corning). Cells in the seed group are grown in ExCeII 302 medium with G418 and anti-agglomerating agent at 37 ° C, 5% CO 2. Bioreactors are inoculated with 0.6x10® cells / mL suspended in 3 L Excell 302 medium without G418 and without anti-caking agent. Viable cell / cell numbers are monitored daily by Casy and ViCeII count. At 50 hours, 2,000 ml of ExCeII 302 medium is supplemented and after 92 hours a temperature drop from 37 ° C to 32 ° C is performed. The cell culture supernatant is collected after 164 hours and subjected to purification as described in Section i).

i. Purification of Individual Anti-RSV Antibodies and Polyclonal Anti-RSV Antibodies

Antibodies expressed as described in Section g.g-2 and h, all of the IgGI isotype, were affinity purified using a MabSeIect SuRe (Protein A) column. Individual antibodies interacted with immobilized Protein A at pH 7.4, while contaminating proteins were washed from the column. Bound antibodies were subsequently eluted from the column by reducing the pH to 2.7. Antibody-containing fractions determined from 280 nm absorbance measurements were pooled and buffer exchanged using a G-25 column in 5 mM sodium acetate, 150 mM NaCl, pH 5 and stored at -20 ° C. ° C.

j. In vitro neutralization assays j-1 Preparation of live RSV for in vitro use Human laryngeal epithelial HEp-2 cells (ATCC CLL-23) were seeded in 175 cm2 flasks at 1x107 cells / vial. The cells were infected with RSV Long (ATTCC VR-26 number), RSV A2 (Advanced Biotechnologies Inc., ATCC VR-1540 number), RSV B1 (ATCC VR-1400 number) or RSV B Wash / 18537 (Advanced Biotechnologies). Inc., ATCC Number VR-1580) in 3 ml serum free medium at a rate of 0.1 pfu / cell. Cells were infected for 2 hours at 37 ° C; 5% CO 2 followed by addition of 37 mL of complete MEM medium. Cells were incubated until cytopathic effects became visible. The cells were scraped off and the media and cells were sonicated for 20 seconds and aliquoted, instantaneously frozen in liquid nitrogen and stored at -80 ° C.

j-2 Plate Reduction Neutralization Test (PRNT)

HEp-2 cells were seeded in 96 well culture dishes

2x104 cells / well, and incubated overnight at 37 ° C; 5% CO2. The substances were diluted in serum free MEM and preincubated with RSV in the absence or presence of complement (Rabbit complement sera, Sigma) for 30 minutes at 37 ° C. This mixture was applied to the HEp-2 cell monolayer and incubated for 24-72 hours at 37 ° C; 5% CO2. Cells were fixed with 80% acetone; -20% PBS for 20 minutes. After washing, biotinylated goat anti-RSV antibody (AdB Serotec) was added (1,200) in PBS with 1% BSA and incubated for 1 hour at room temperature. After washing 25 HRP-avidin was added and allowed to incubate for 30 minutes. The plates were developed by incubation with 3-amino-9-ethylcarbazole substrate (AEC) until the plates were visible by microscopy, for example for 25 minutes (RSV LOng) or 45 minutes (RSV BI). Plates were counted in a Biorreator (Bio-Syz GmbH). EC 50 values (effective concentrations required to induce a 50% reduction in plate number) were calculated, where applicable, to allow for a power comparison. j- 3 Fusion Inhibition Assay

The fusion inhibition assay was essentially performed as the plaque reduction neutralization assay except that RSV was allowed to infect prior to the addition of test substances. In practice, the virus was added to the serum free medium to the HEp-2 cell monolayer for 1.5 h. The supernatants were removed and test substances were added in complete MEM medium with or without complement (Rabbit complement sera, Sigma). Plates were incubated overnight and processed as described above for the plaque reduction neutralization assay.

j- 4 Micronutralization test

In addition to the PRNT and fusion inhibition assay as described in Sections j-2 and j-3, a microneutralization assay based on RSV protein detection was employed for the determination of RSV neutralization and fusion inhibition.

For the neutralization test, the test substances were diluted in serum free MEM and allowed to preincubate with RSV in the absence or presence of complement (Rabbit complement sera, Sigma) in 96-well culture plates for 30 minutes. at room temperature. Trypsinized HEp-2 cells were added at 1.5x10 4 cells / well, and incubated for 2-3 days at 37 ° C; 5% CO2. The cells were washed and fixed with 80% acetone; 20% PBS for 15 minutes at 4 ° C and dried. The plates were then blocked with PBS with 0.5% gelatin for 30 minutes at room temperature and stained with a pool of 25 murine RSV protein (NCL-RSV3), Novocastra) diluted 1: 200 in PBS with 0.5% gelatin and Tween-20 at

0.5% for 2 hours at room temperature. After washing, Polyclonal Rabbit Anti-mouse Immunoglobulin HRP Conjugate (P0260; DakiCytomation), diluted 1: 1000 in PBS with 0.5% gelatin and 0.5% Tween-20 was added and incubated. for 2 hours at room temperature. The plates were washed and developed by addition of orthophenylendiamine. The reaction was terminated by addition of H2SO4 and the plates were read on an ELISA plate reading at 490 nm.

The fusion inhibition assay was essentially performed as the microneutralization test except that the virus was added and incubated for 1.5 h at 37 ° C; 5% CO 2 before test substances, 5 diluted in complete MEM have been added. The plates were incubated for 2-3 days at 37 ° C; 5% CO2, developed as described above.

k. In vivo protection tests

k-1 Mouse challenge model Female MBALB / c mice, 7 to 8 weeks old, were innocuous

intraperitoneally with 0.2 mL of antibody preparation on day 1 of the study. Placebo-treated mice were similarly inoculated i.p. with 0.1 mL PBS buffer. On day 0 of the study, mice were anesthetized using inhaled isofluorane and intranasally inoculated with 10'6-10'7 pfu of the RSV A2 strain at 50 μί or with cell lysate (mock inoculum). The animals were left for 30 seconds aspirating the inoculum while standing until fully recovered from anesthesia.

Five days after the challenge, the mice were killed with an overdose of sodium pentobarbitone. At postmortem, blood was obtained by exsanguination of the axillary vessels to prepare the sera. The lungs were removed and homogenized in 2.5 mL sterile buffer. Lung homogenates were centrifuged to sediment sand and cell debris and supernatants were aliquoted and stored at -70 ° C.

In a long-term version of the challenge model, groups of animals were killed at different times, ie, 5, 27, and 69 days after challenge, and serum sample homogenates were prepared as described above. In addition, separate groups of 30 animals that were killed at the same time were used for bronchoalveolar lavage (BAL) and histopathology samples. In summary, the airways were cannulated and flushed with 1 mL of saline. The total number of cells present in the BAL was determined by light microscopy. BAL cell cytosine preparations were stained with hematoxylin and eosin and differential cell counts made using immersion microscopy. After lavage, the lungs were fixed for histopathology. Fixed tissue samples 5 were prepared by inflating the lungs with buffered formalin, and the fixed tissue was embedded in paraffin blocks for processing and staining with hematoxylin and eosin by standard methods. Lung pathology scores were determined as the sum of the severity score multiplied by the prevalence score for each 3 bosοί 0 bos of the lung (Table 5a). The maximum lung pathology score for a mouse is thus 36.

Table 5a. The lung pathology score system uses

for mouse challenge studies.

Severity Prevalence 0 Normal 0 Normal 1 perivascular and peri- cell infiltration <25% of the bronchial specimen <3 cells thick 2 perivascular and peri- cell infiltration 25 to 50% of the abbreviated 4 to 10 cell thickness shows 3 infiltration of perivascular cells and peri- 3 3 51-75% of bronchial samples> 10 cells thickness tr 4> 75% of sample Virus load was initially determined by quantification

of RSV RNA copy number in lung samples using reverse transcriptase (RT) -PCR. RNA was extracted from the lung homogenate samples using the MagNA Pure LC Total Nucleic Acid Kit (Roche Diagnostics) extraction system according to the manufacturer's instructions. RSV RNA detection was performed by real-time single-tube RP-PCR using the LightCycIer instrument and reagents (Roche Diagnostics) with subtype A RSV gene specific primers and fluorophore probes as described by Whiley et al. . (J, Clinical Microbiol. 2002, 40: 4418-22). Samples with known RSV RNA copy numbers were similarly analyzed to derive a standard curve.

Subsequently, the RSV RNA copy number in the 5 lung samples was determined during quantitative reverse transcriptase (RT) -PCR. RNA was extracted from lung homogenate samples using the RNeasy mini-kit (Qiagen) according to the manufacturer's instructions. RSV RNA detection was performed using the One-Step SuperScript IN Platinum RT Invitrogen RT PCR System (Invitrogen) with 10 primers and fluorophore-labeled probes for the RSV N gene subtype A as described in Table 5b, below, down, beneath, underneath, downwards, downhill. Samples with RSV RNA copy numbers were similarly analyzed to derive a standard curve.

Table 5b. Subtype A-specific RSV primers and 'RT-PCR probe'.

Name Sequence 5 '- 3' Advance RSV-A adv- CAA CAA CAA AGA TCA ACT TCT GTC ATC Reverse GCA CAT CAT CAT AAT TAG GAG TAT CAA T RSA Probe 6-FAM-CA CCA TCC AAC GGA GCA CAG GAG Os Different cytokine and chemokine levels in lung tissue samples were determined by a commercial mutiplex immunoassay in Rules-Based Medicine (Austin, TX) using its rodent multianalysis profile (MAP).

k-2 Cotton Mouse Challenge Template

Female 6- to 8-week-old cotton mice (Sigmodon hispidus) are inoculated peritoneally with 0.5 mL of the antibody preparation or placebo (PBS) on day 1 of the study. Twenty-four hours later, the animals are lightly anesthetized with isofluorane and an intranasal challenge of 10'6-10'7 pfu of RSV strain 42 or control medium (mock inoculum) was given. A total volume of 100 μί of inoculum is administered and evenly distributed to both nostrils. After completing the intranasai challenge, each animal is held upright for a minimum of 30 seconds to allow full inhalation of the inoculum. Five days after challenge, animals are killed by lethal intraperitoneal injection of pentobarbitone and exsanguinated by cardiac puncture. Serum samples are obtained and frozen at -80 ° C and each animal is dissected under aseptic conditions 5 for removal of lungs and nasal tissue. Tissue samples are homogenized and the supernatants stored in aliquots at -80 ° C.

Virus load in tissue samples is determined by quantifying RSV RNA copy number by a Taq-Man real-time assay based on the method of Van Elden et al. (J Clin Microbiol. 10 2003, 41 (9): 4378-4381). In summary, RNA is extracted from lung homogenate samples using the Rneasy (Qiagen) method according to the manufacturer's instructions. RNA is extracted and reverse transcribed into cDNA and subsequently PCR amplified using the One-Step Quantitative RT-PCR System (Invitrogen) with primers and 15 labeled probes specific for subtype A RSV N gene with RSV concentrations Known parameters are similarly analyzed to derive a standard curve.

k- 3 Pharmacokinetic study in mice 7 to 8 week old female BALB / c mice were inoculated intraperitoneally with 0.2 ml antibody preparation on day 0. Serum samples were taken from the orbital plexus at multiple times (0 hour, 4 hours). hours, days 3, 6, 9, 13, 16, 21, 24, and 29) after antibody treatment. Mice were sacrificed and homogenized in 1.5 ml buffer using a tissue lysis buffer (tissuelyzer) zer (Qiagen). The lung homogenates were thereafter centrifuged to pellet the cell debris and the supernatants were stored at -70 ° C. Human antibody levels present in serum samples and lung homogenates were measured using human IgGI kappa ELISA.

EXAMPLE 2

In the present Example, the isolation, screening, selection and bank formation of clones containing cognate Vh and Vl pairs expressed as life-size antibodies with anti-RSV specificity are illustrated.

Donors

A total of 89 donors were recruited from employees and relatives who were hospitalized in the Department of Pediatrics at Hvidovre Hospital (Denmark) during the RSV season. An initial 18 mL blood sample was taken, CD19 + B cells were purified (Example

1, Section a) and screened for anti-RSV antibodies using ELISpot (Example 1, Section b) and plasma cell frequency was determined by FACS analysis.

Eleven donors were detected as positive in the selection of initial blood samples and a second 450 mL blood sample was collected from ten of these. Plasma blasts were single cells chosen according to Example 1, Section a. ELISpot was performed on a fraction of CD19 positive B cells.

Four donors with ELISpot frequencies in the second blood donation between 0.2 and 0.6% of RSV-specific plasma cells (IgG 'and IgA +) of the total plasma cell population were identified. These frequencies were considered high enough to bind repertoires of cognate Vh and Vl pairs.

Isolation of Vh and Vl encoder pairs

Nucleic acids encoding antibody repertoires were isolated from selected single-cell plasma cells of the five donors by multiplex overlap-extension RP-PCR (E25 x 1, section c). Multiplex overlap-extension RT-PCR creates a physical link between the heavy chain variable region (Vh) gene fragment and the life-size light chain (LC). The protocol was designed to amplify antibody genes from all Vh gene families and the kappa light chain using two primer sets, one for Vh amplification and one for LC amplification. Following reverse transcription and multiplex overlap-extension PCR, the ligated sequences were subjected to a second PCT amplification with a first set of nested primers.

Each donor was processed individually, and 1480 to 2450 overlap products were generated by multiplex overlap extension RT-PCR. The generated collection of cognate Vh and V15 coding pairs from each donor were pooled and inserted into a mammalian IgG expression vector (Figure 3) as described in Example 1 section d). The generated repertoires were transformed into E. Co

li, and consolidated into twelve 384-well master plates and stored. The repertoires consist of between 1x106 and 3.6x10® clones per donor.

Selection

IgG antibody-containing supernatants were obtained from CHO cells transiently transfected with DNA prepared from master plate bacterial clones. Supernatants were selected as described in Example 1, section e. Approximately 600 primary ranges 15 were sequenced and aligned. Most fell into groups of two or more members, but there were also clones that were isolated only once, the so-called singletons. Representative clones of each group and singletons were subjected to validation studies as described in Example 1, section e). Several primary ranges were excluded for further characterization due to unwanted sequence characteristics such as mismatched cysteines, non-conservative mutations, which are potential PCR errors, multiple codon insertions and / or deletions, and truncations.

A total of 85 unique clones passed validation. These are summarized in Table 6. Each clone number specified a particular pair of Vh and Vl. The IGHV and IGKV gene family is indicated for ca

clone and specifies the frame regions (FR) of the selected clones. The amino acid sequence of the complementarity determining regions (CDRs) of an antibody expressed from each clone is shown, wherein 30 CDRH1, CRH2, CDRH3 indicate heavy chain CDR regions 1, 2 and 3 and CDRL1, CDRL2 and CDRL3 indicate the light chain CDR regions 1, 2 and 3. The sequence of chains, heavy and light, complete variables can be established from the information in Table 6.

Further details for the individual columns in Table 6 are given below.

The names of the IGHV and IGKV gene families have been designated

according to the official nomenclature HUGO / IMGT (IMGT; Lefranc & Lefranc, 2001, The Immunoglobulin FactsBook, Academic Press). Numbering and alignments are according to Chothia (Al-Lazikani et al. 1997 J. Mol. Biol. 273: 927-48). Clone 809 has a two-codon 5 'insert into CDRH1, which probably translates into an extended CDR loop. Clone 831 has a codon deletion at position 31 in CDRH1.

The "AG" column indicates the RSV-associated antigen recognized by the antibody produced from the named clone as determined by ELISA, FLISA and / or Biacore. "+" indicates that the clone binds to RSV particles and / or RSV infected cells, but that of the antigen has not been identified.

The "Epitope" column indicates the antigenic site or epitope recognized by the antibody produced from the designated clone (see Table 4 and below). "U" indicates that the epitope is unknown. UCI and UCII refer to the unknown group I and II. Antibodies belonging to these groups have similar reactivity profiles, but are currently not viewed as assigned to a particular epitope. Some antibodies recognize complex epitopes, such as A&C. The epitopes indicated in () were only identified by ELISA. Table 6: Sequence Summary and Specificity of Each Unique Validated Clone IGHV CDRH1 CDRH2 CDRH3 IGKV CDRL1 CDRL2 CDRL3 Ag Epitope Clone Gene 3 3 5 6 9 0 0 gene 2 3 3 5 89 9 1ab2345 012abc3456789012345 234567890abcdefghijklmnodel 456787834434645634140645634123 -YDWS NIN-YRGNTNYNPSLKS CARDVGYGGGQYFAM-DVW 3-11 RASQSVNS-HlA NTFNRVT CQQRSNWPPALTF F UCI 736 3-30 T-YGMH FIRY-DGSTQYYYYYYYE YALT RITP-MFDaNYAQKFQG CARRGAVALVPAAEDPYYYGM- 2-28 RSSQSLLHS-NGNNYLD LASNRAS CMQSLQT --- PTF G Central Domain DWV 744 1-2 G-YYMH WINT-SSGGTNYAQKFQSRWYYYYYYYYYYYYYYYYYYYYYYYYYYYY RDP -11 D-YYMS YINR-GGTTIYYADSVKG CARGLILALPTATVELGAF- --DIW 1-39 RASQSITG-YLN ATSTLQS CQQSYNT --- LTF G Preserved 794 1-18 N-YGLN WINA-YNDNTYYSPSLQG CARSYRSQTDILTGRYKGPGDVFDN 1-12 RASEGISS --- WLA AASTLQS CQQTNSFP-YF7YYYY HSGTTYYNPSLKS CARDVDDFPVWGMNRYL --- ALW 3-20 RASQSVSSS --- YLA GASTGAT CQQYGRTP-YTF F UCI S 796 3-30 H-FGMH IISY-DGNNVHYADSVKY-29JKF-EYRJJF-29RF R-FGIS WISA-DNGNTYYAQNFQD CVRGGWTNRVYYYYGM-DWV 1-9 RASQGISS-YLA AASUQS CQQVDTYP-LTF G GCRRA 798 7-4-1 S-YVMN WINT-NTGDPAYAQDFT GWFQRQF-FFYRQF-FWRQY 799 3-30 N-YGMH VISY-DGRNKYFADSVKG CARGSVQVWLHLGLF-DNW 1-5 RASQSVSS --- WVA EASNLES CQQYHSYSG-YTF FU 800 3-33 D-YGMN VIWH-DGSNKNYLDSVKG CARTPYFS-FQFDS-FQS F1 801 3-33 S-YAMH VIYY-EGSNEYYADSVKG CARKWLGM-DFW 2-28 RSSQSLLNS-NGFNYVD LGSNRAS CMQALETP-LTF F F1 802 3-48 S-YEMN YIGT-GGSDIYYGDSVKG CARARPGYKV-DFW 1-9 RASQGISS-YLA VASILES CQQSKSFP-YF FUY-SGS-YF 3-20 RASQTVSSS --- YLV GASTRAT CQQYGGSG-LTF FUS 804 3-64 N-YAMH ATST-DGGSTYYADSLKG CARRFWGFGNFF-DYW 3-20 RASQSVSSG --- YLA GASGRAT CQQYFGSP-YTF F1-YYY FY 80Y CAREGHHSGSGDYYSFF-DYW 1-39 RASQGINT --- YLN AASSLQS CQQSANSP-HTF F (F1) 806 5-51 S-YWIG IVYP-GDSDTTYSPSFQG CVRRGGFCTATGCYAGHWF --- 3-20 RASQSISSG --- YQQ GASH 2 -70 T- RID-WDDDKYYSTSLKT CARIVFHTSGGYYNPYM-DVW 1-39 TYPE-YLS TASSLQS CQHSYNTP-YTF F (F1) TRMSVS 809 5-51 FVSTWIG IINP-ADSDTRYSPSFQG YF-YF 810 1-69 N-YAIN I RIIP-VFDTTN YAQKFQG CLRGSTRGWDTDGF-DIW 1D-17 RASQGISN --- YLV AASSLQS CLQHNISP-YTF FM 811 1-46 N-YYIH VINP-NGGSTTSAQKFQD CARQRSVTGGFDAWLLIPYYYYYYYYYYYE YAQTFQG CARVFREFSTSTLDPYYF --- DYW 3-20 RASQSVSSS --- YIA AASRRAT CQHYGNSL-FTF F F1 813 5-51 S-YWIG IIYP-GDSDTRNSPSFQG CVRQGGYYDRNGYHEKYAF --- DIWES F1F YES 814 I 3-30-3 D-YAMH VISY-DGANEYYAESVKG CARAGRSSMNEEVIMYF-DNW 1-5 RASQSIGS-RLA dassles CQQYNRDSP-WTF G Conserved 816 3-23 817 3-30 818 2-70 819 4-304 822 5-51 823 4-b 824 4-59 825 1-18 827 1-24 828 1-3 829 2-70 830 1-18 831 1-3 833 3-30 834 1-18 835 1-18 836 4-b 838 3- 30 839 3-30 841 1-18 842 1-18 843 1-18 845 1-18 846 4-302 848 4-61 849 3-73 850 1-3 851 1-18 852 1-69 853 5-51 855 1-18 856 1-18 857 3-23 τ-YAivrr T-HGMH A

GRVGVS

GADYYW

s

N-SWIG

SG

HFWG

N-YYWG

S-NGLS

A-LSKH

T-NGLH

RN

RMSVS

T-YGVS

-YAMH Y-IGMH T-YGLN S-YGFS SGHYWG T-FGMH S-YGLH S-FGIS R-YGIS N-SGVS S-YGIS SGGYSW S

SDKNYW

s

G-STMH

T-YTLH

S-LGFS

G-YTIH

N-YWIG

N-YAFS

N-YGFS

S-YAMN

VIRA-SGDSEIYADSVRG

ItSL-DGIKTHYADSVKG

RID-WDDDKAFRTSLKT

Fir-DSGSTYYNPSLRS

11YP-GDSTTTYTPSFQG

SIF-HSGTTFHNPSLKS

HIY-FGGNTNYNPSLQS WISA-SSGNKKYAPKFQG FFDP-EDGDTGYAQKFQG LINA-GNGDTRFSQKFQG

RID-WDDDKFYNTSLQT

WISA-YNGNTYYLQKLQG

WINV-GNGQTKYSQRFQG

AISY-DGSNKQYADSVKG

VWSA-HNGNTYYAEKFHD

WSSV-YNGDTNYAQKFHG

SIY-DSGNTYYTPSLKS

VtSY-DGNKKYYADSVKG

EISY-DGGSKFYTDSVKG

WISA-YNGNTDYAQRLQD

WISA-YNGNTYYAQNLQG

WISA-YNGNTYYRQSLQD

W1GT-DNGNTYYAQKFQG

YIY-HSGSTYYNPSLKS

RLY-PSGNTDYHPSLKS

RIRSKANSYATEYAASVKG

LINA-ANGHTKYSQRFQG

WTSA-HNGNTYYAEEFQD

RLVP-SLNIPNYAQKFQG

VIFP-ADSDARYSPSFQG

WISG-SNGNTYYAEKFQG

WISA-YNGWTYYAQNLQG

GISG-SGGSTYYGDSVKG

CANIGQRRYCSGDHCYGHF — DYW CAKDHIGGTNAYFEWTVPF — DGW

CARTQVFASGGYYLYYL-DHW

CARDLGYGGNSYSHSYYYGL—

DVW

CARQGRGF-GLW

-DHW

CARVHGGGF

CARDSSNWPAGY

-EDW

CAKDGGTYVPYSDAF-DFW

CATVAAAGNF-DNW

CARIAITMVRNPF-DIW

CARD Gl YDSSGYYLYYF-DYW

CARDRVGGSSSEVLSRAKNYGL

DVW

CARRASQYGEVYGNYF-DYW

CAKDDFGNSNGVFFMSRV — AFW CVRGFNEQQLVPGLSFWF — DYW CARDRNWLLPAAPFGGM — DVW

CARGSPGDAF

-DIW

CAAQTPYFNESSGLV-PDW

CARDLGDGYTAWGWF-DPW

CTRDESMLRGVTEGFGPI — DYW

CV1SFDSTIAAAEYF-DYW

CAREGHYSGSSSYQRDDAF — DIW CARGGTIEATPEREYYYYGM — DVW

CASRSFYGDY

-WW

CAKEGSGWYF

-ESW

CTRHVGEMSTIWWYF-DLW

CAKSGSHY GEVYGAYF-DYW

CARDRGPGYSDSSFYVF-DYW

CTRAPRGSTASHLLF-DYW

CARPKYYFDSSGQFSEMYYF — DFW

CARDLLRSTYF-DYW

CARDGNTAGVDMWSRDGF — DIW

CAKEPWIDIWASVISPYYYDGMDV

W

2-28

3-15

1-39

3-11 1D-33 1D-33

1D13

4-1 1-39 1-5

1-39

1-5

1-5

1-12

1-12

1-9

1-12

1-27

3-20

4-1 1-5 1-16 1-9

3-20

1-5

1-39

1-5

2-24 1D-33

3-20 1D-12 2-40

2-28

RSSQSLLHS-DGRYYVD WASQTIGG-NLA

RASQTIAS-YVN

RASQSVSS-SLA

QASQDITY-YLS

QASQDIGD-SLN

RPSQDISS — ALA KSSQSVLYNSNNKNYLA

RASQFISS-YLH

RASQSIGS — WLA

RASQSIAS-YLN

RASQSVTS-ELA

RASQNIYN — WLA

RANQDIDN-YLA

RASQGISK-RLA

RASQGISS-YLA

RASQGIGT — WLA

RASQGI SN-YLA

RASQSVGGR — SLA RSSQSVLYSSNNKNYLA

RASQTISN-SLA

RASQGISN-YLA

RASQGISS-YLA

RASQSVSSS — YLA

RASQGISA — WlA

RASQSISS-YLN RASQNIYN-WLA RSSQSLVNS-DGNTYLS QASQDVSY-YLN RASQSVSSN-YLA

RASQAISN-WLA

RSSQSLLDSNDGNTYLD

RSSQSLLHR-NEYNYLD

LASNRAS

GASTRAT

AASNLQS

DASYRVT

DVSNLER

DASNLET

GASTLDY

LASTREY

AASTLQS

KESNLES

AASSLHS

KASSLES

DASTLES

GASKLQT

GASSLQH

AASTLQS

AASRLQS

AASTLQS

DASNRAT

WASTRAS

KASTLES

TTSURS

AASUQS

GASSRAT

DASTLAS

AASSLQS

DASSLES

QISKRFS

DTSNLVT

GASSRAA

AASSLQS

TFSYRAS

WGSNRAS

CMQGLHTP-WTF CQQYKNW — YTF

CQQSYSYRA-LTF

CQQRSNWPPGLTF

CQQYDFLP-YTF

CQHYVNLPPSFTF

CQQFNTYP-FTF CQQYYQTP-LTF CQQS YTNP-YTF CQQYKND — WTF

CQHSYSTR-FTF

CQQYNSFP-YTF

CQQYNSLS-PTF

CQQAKSFP-FTF

CQQADSFP-FTF

CQQLNSYP-R7F

CQQAYSFP-RTF

CQKYNSAP-QTF

CQQYGSPP-WTF

CQQFHSTP-RTF

CQQYNSFS-FTF

CQQYHSFP-YTF

CQQLNTYP-LTF

CQQYGSSP-FTF

CQQYRSYS-YTF

CQQSYSTP-YTF

CQQYNIYS-PTF

CMQATQFP-FTF

CLQYHYLP-YTF

CQQYGNSP-LTF

CQQADTFP-FTF

CMQRIEFP-YTF

CMQUQTP-RTF

G Conserved FA / ll FB / I / F1 A / ll FFUU + FA / ll * (F1 & C) FA / ll * (UCI) F (A & COIV * + A&C FU (F1) G GCRRA FA / ll G Central domain G GCRRA G GCRR F (A / ll) G Conserved G GCRRA G GCRRA G GCRRA G GCRRA G GCRRA FUFUFUF (ΑΛΙ) G GCRRA FUG Central Domain G GCRRA G GCRRA F F1 CD

The 858 1-69 G-YTIS RWP-TLGFPNYAQKFQG CARMNLGSHSGRPGF-DMW 1D-33 QASQDISN-YLN DATKLET CQHFANLP-YTF FB / I / F1 859 3-33 LTF G Conserved GYW 861 3-30 S-YGMH FIWN-DGSNKYYADSVKG CVKDEVYDSSGYYLYYF-DSW 1-39 RASQIIAS-YLN AASSLQS CQQSYSTPI-FTF F1 863 3-23 S-YTMS SISA-STVLGYYYYYYYYYYYYYY CQQRSDW --- LTF FA / ll DLW 866 1-18 T-YGIS WISA-DNGNTYYAQKFQG CVRGGTYSSDVEYYYYGM --- DVW 1-9 RASQGISI --- YLA AASUQT CQQLNIYP-LTF G GCRRA 867 1-69 R-YTIH RGYP DSW 1D-33 QASQDINN-YLN DATDLET CQHFANLP-YTF F (F1) 868 4-b NA-SIH-HSGSAYYNSSLKS CARDTILTFGEPHWF DPW 3-15 RASQSIKN-NLA GASARAT CQEYNNWPL-LTF YDYYYYG GYARYYGYYLY -DSW 3-20 RASQSLSDN --- YLA GASSRPT CQQYGTTP-ITF G Conserved 870 4-59 N-YYWS EIS --- NTWSTNYNPSLKS CARGLFYDSGGYYLFYF-QHW 1n39 SCALES-YLN AASSLQS CQQSYSTPI-YTF F (F1> 871 3-33 N-YGMH VIWY-DDSNKQYYYYYYYYYYYYYYYYYYYE DGNIKYSADSVKG CHGEGYSTSWLGTAAL DYW-FLA-1-27 RASQGIRN AASTLQS CQKYNSAP Conserved G-WTF A-3-23 879 YAMS AISG-GGGTTYYADSVKG CAKTRGYSYTWGDAF-DLW 3-15 RASQSVÍS NLA-GASTRAT CQQYNNWP QFT-FU 2-5 880 LVD-TS-WDDDRRYRPSLKS CAHSAYYTSSGYYLQYF --- HHW 1-39 RASQTIAS-YVN AASSLQS CQQSYSFP-YTF F UCII KLGVG 881 3-48 S-YEMT HIGN-SGSMIYYADSVKG CARSDYYDSSGYYLLYL --- DSW 1-39 FtASQTIAS-YVN AASNLQS NF-8 LTF-8 UF FAMH YINA-VNGNTQYSQKFQG CARNNGGSAIIF-YYW 1-39 RSSQTISV --- FLN AASSLHS CQESFSS --- STF FU 885 4-b SN-SMH-HSGSSYYKPSLKS CARDLWVTDISIKNYF-DPW 3-11 RASQSVTK YLA-YF 3-30 S-YGMH VISN-DGSNKYYADSVKG CAKTTDQRLLVDWF-DPW 3-15 RA SQSVSS-NLA SASTRAT CQQYNMWPP-WTT = FA / ll 887 2-70 TS- RID-WDDDKYYSTSLKT CARTLVYAPDS YYLYYF-DYW 1-39 RASQTIAS-YVN AASRLQS CQQSYSIP-WTF YYYYYYYYYYYYYYE 2-28 RSSQSLLRT-NGYNYLD LGSIRAS CMQSLQTS-ITF G GCRR G 889 1-18 T-YGIS WISA-YNGNTFYAQRLQG CARDLRMLPGGLPTRRGM --- DVW 1-5 RASQSISS-WLA KASS LES CQQYNSYP GF-YP-8Y46 SGGSTTYAQTFQD CARGIREGGVSVEDWMLVYSWF- 1-39 RASQNIRT-FIN AASKLES CQQGHSTP-YTF G Conserved DPW 891 3-30 S-YTMH WSY-DGNHNDVADSVKG CVRAPGSMGL-DVW 2-28 RSSQYNHRF-NG7NGHHH CAPLGGPTPF-DYW 1-17 RAGQGIRN-DLG GASUQS CLQHNSYP-WTF + U 893 3-30 I-YGMH VISY-DGAKKFYANSVKG CATASTYFYDSR-DYW 2-24 RSSRSLVHS-DGNTYLS KISNRFS CLQATWH-3 LTF33 --- 33 -DGSNIRYADSVRG CARVPFQIWSGLYF-DHW 3-15 RASQSVGN-NLA GASTR AT CQQYDKWP-ETF FC * (UCI) 924 4-b SE-SVH-HSGSTYYNPSLKS CARDRVALGVHYWYF-DlW 3-15 RASQSVSS-HLA GASTRAT Central Dome YYWG 955 146 D-YCMHYDQD-YCMHYDDD-YCMHYF RASRSITS-WLA KASSLQS CQQYNSYP-LTF SH A2 aa42-64 Further experiments confirmed binding to the epitope with an asterisk. The top-down amino acid sequences in the column designated CDRH1 are shown in the same order in SEQ ID NOs: 201-285.

The top-down amino acid sequences in the column designated CDRH2 are shown in the same order in SEQ ID NOs: 286- 370.

The top-down amino acid sequences in the column labeled CDRH3 are shown in the same order in SEQ ID NOs: 371-455.

Top-down amino acid sequences in the column

names CDRL1 are shown in the same order in SEQ ID NOs: 456-540.

The top-down amino acid sequences in the column labeled CDRL2 are shown in the same order in SEQ ID NOs: 541-625.

The top-down amino acid sequences in the column labeled CDRL3 are shown in the same order in SEQ ID NOs: 626-710.

Antigen specificity characterization During validation, the antigen specificity of clones was

determined to the same degree by binding of soluble viral particles, protein G and protein F, as well as fragments of protein G.

For clones with anti-F reactivity, the specificity of the expressed individual antibodies of the clones was subsequently evaluated to determine the antigenic site, and, if possible, the epitope bound by the individual clones (see Example 1, Section g-4). Figure 4 illustrates the characterization of antibody epitope specificity obtained from clone 801 using Biacore analysis. Analysis shows that when protein F is blocked by 133-1h or Palivizumab (antigen site C and II, respectively) prior to injection of 801 antibody into the Biacore cell, a high degree of 801 antibody binding can be detected. Binding of uncompeted 801 antibody is somewhat reduced compared to binding of competing 801 antibody. The reduction is, however, so low that it is more likely to be due to steric hindrance than direct competition to the binding site. Blockade of protein F with antibody 9c5 (F1 antigenic site) prior to injection of antibody 801 into the Biacore cell shows an almost complete inhibition of binding of antibody 801 to protein F1 and therefore concluded that antibody 801 binds to protein F at F1 site, or very close to it.

For clones with anti-G reactivity, the specificity of the individual antibodies expressed from the clones was subsequently evaluated to determine whether the antibody binds to the 10G protein central domain, conserved region, or GCRR, and also whether the epitope is conserved or subtype-specific. This was done by ELISA and / or FLISA using the following G protein fragments:

G (B); residues 66-292 from strain RSV 18537 (expressed in CHDO DE DG44 cells)

Fragment G (B): Residue 127-203 from RSV strain 18537 (expressed in E. coli).

GCRR A: RSV Long strain residues 171-187 (synthesized with selectively formed cysteine bridges).

GCRR B: RSV strain 18537 residues 171-187 (synthesized with selectively formed cysteine bridges).

Preserved G: Residues 164-176.

Additional epitope analyzes were also performed on anti-G reactive clones by competition assays as described in Example 1, Section g-4.

Also, one of the clones identified in a procedure of

as described in Example 1, Section e, produces a SH-specific antibody. Additionally, several clones bind to one or more of the RSV strains tested, but the antigen has not been determined.

Antigen specificity data for all validated clones are summarized in Table 6. None of the validated clones bind to human laryngeal epithelial cells and also none of the G-specific clones tested (793, 816, 835, 841, 853, 855 , 856, and 888) binds a human fractalcin (CX3CL1).

Binding kinetics characterization

Binding affinity for recombinant RSV antigens was determined by surface plasmon resonance for various antibody clones. The analysis was performed with Fab fragments prepared by enzymatic cleavage with K 0 values in the picomolar to nanomolar range are shown in Table 7. The commercially available Palivizumab (Synagis) derived Fab fragments were similarly analyzed for reference.

Table 7: Kinetic Binding Constants and Clone Affinities

implemented. Fab clone kon koff t '/ 2 Kd (antigen) (105M'1 s'1) (10'5 1 / s) (min) (pM) 735 (F) 4.07 9.18 130 226 810 (F ) 17.40 34.80 33 200 818 (F) 1.92 2.20 530 115 817 (F) 0.92 7.54 150 820 819 (F) 3.56 4.99 230 140 825 (F) 7 , 72 15.00 77 195 858 (F) 4.97 0.34 3400 7 831 (F) 3.72 42 28 1130 796 (G) 8.33 40.3 28.67 480 811 (G) 4.98 17.1 68 340 816 (G) 20.20 17.80 65 90 838 (G) 2.64 5.06 230 190 853 (G) 17.7 140 8.25 790 859 (G) 3.8 4, 63 250 120 Synagis (F) 2.00 75,70 15 3780 Generation of a clone cell bank expressing an individual antibody

A subset of 47 pairs of Vh and Vl cogna encoders

unique, corresponding to clones N0

735, 736, 744, 793, 795, 796, 799, 800, 801, 804, 810, 811, 812, 814, 816, 817, 818, 819, 824, 825, 827, 828, 829, 830, 831, 835, 838, 841, 853, 855, 856, 857, 858, 859, 861, 863, 868, 870, 871, 880, 881, 884, 885, 886,

888, 894 and 955 in the Table were selected to generate individual expression cell lines, each expressing a unique antibody of

a sequence of simple Vh and Vl genes. The entire sequences (DNA and

deduced amino acids) from 44 selected clones (except those identified

828, 885, and 955) are shown in SEQ ID NOs: 176;

The 44 clones are characterized by producing the following

VH sequences, which are set forth in SEQ ID NOs: 1-44:

Clone No. 735:

QVQLQESGPGLVKPSETLSLTCTVSNGAIGDYDWSWIRQSPGKGLEWIGNI NYRGNTNYNPSLKSRVTMSLRTSTMQFSLKLSSATAADTAVYYCARDVGY GGGQYFAMDVWSPGTTVTVSS Clone No. 736:

QVQLVESGGGWQPGGSLRLSCTASGFTFSTYGMHWVRQAPGKGLEWV AFIRYDGSTQDYVDSVKGRFTISRDNSKNMVYVQMNSLRVEDTAVYYCAK D YYG S RS YS VTYYYG M D VWGQGTTV: 7

QVQLVQSGAEVKKPGASVKVSCKASGYTFSGYYMHWVRQAPGQGLEWM GWINTSSGGTNYAQKFQGRVTMTRDTSISTAHMELRRLRSDDTAVYYCAR EDGTMGTNSWYGWFDPWGQGTLVTV No. 7

QVQLVESGGGLVKPGGSLRLSCAASGFPFGDYYMSWIRQAPGKGLEWVA YINRGGTTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAGDTALYYCARGLI LALPTATVELGAFDIWGQGTMVTVSS Clone No. 795:

QVQ LQES G PG LVKPSQTLS LT CTVSG AS ISSG DYYW SWIRQS P RKG L EWI GYIFHSGTTYYNPSLKSRAVISLDTSKNQFSLRLTSVTAADTAVYYCARDVD DFPVWGMNRYLALWGRGTLVTVSS Clone No. 7

QVQLVESGGGWQPGRSLRLSCAASGFSFSHFGMHWVRQVPGKGLEWV AIISYDGNNVHYADSVKGRFTISRDNSKNTLFLQMNSLRDDDTGVYYCAKD DV ATDLAA YYYFDVWGRGTLVTV No. 7:

QVQLVESGGGVVQPGRSLKLSCEASGFNFNNYGMHWVRQAPGKGLEWV AVISYDGRNKYFADSVKGRFIISRDDSRNTVFLQMNSLRVEDTAVYYCARG

SVQVWLHLGLFDNWGQGTLVTVSS

Clone No. 800:

QVQLVESGGAWQPGRSLRLSCEVSGFSFSDYGMNWVRQGPGKGLEWV AVIWHDGSNKNYLDSVKGRFTVSRDNSKNTLFLQMNSLRAEDTAVYYCAR TPYEFWSGYYFDFWGQGTLVTVSS Clone No. 80

QVQLVESGGGWQPGRSLRLSCAASGFPFNSYAMHWVRQAPGKGLEWV AVIYYEGSNEYYADSVKGRFTISRDNSKNTLYLQMDSLRAEDTAVYYCARK WLGMDFWGQGTL VTVSS Clone No. 804:

EVQLVESGGGLVRPGGSLRLSCSASGFTFSNYAMHWVRQAPGKRLEYVS ATSTDGGSTYYADSLKGTFTISRDNSKNTLYLQMSSLSTEDTAIYYCARRF WGFGNFFDYWGRGTLVTVSS Clone No. 810:

QVQLVQSGAEVKKSGSSVKVSCRASGGTFGNYAINWVRQAPGQGLEWVG RIIPVFDTTNYAQKFQGRVTITADRSTNTAIMQLSSLRPQDTAMYYCLRGST RGWDTDGFDIWGQGTMV11SS: 8

QVQLVQSGAVVETPGASVKVSCKASGYIFGNYYIHWVRQAPGQGLEWMA VINPNGGSTTSAQKFQDRITVTRDTSTTTVYLEVDNLRSEDTATYYCARQR SVTGGFDAWLLIPDASNTWGQGTMV12SS: Clone No. 8

QVQLVQSGAEMKKPGSSVKVSCKASGGSFSSYSISWVRQAPGRGLEWVG MILPISGTTNYAQTFQGRVIISADTSTSTAYMELTSLTSEDTAVYFCARVFRE FSTSTLDPYYFDYWGQGTLVTVSS Clone No. 814:

QVQLVESGGGWQPGKSVRLSCVGSGFRLMDYAMHWVRQAPGKGLDWV AVISYDGANEYYAESVKGRFTVSRDNSDNTLYLQMKSLRAEDTAVYFCARA GRSSMNEEVIMYFDNWGLGTLVTVSS Clone No. 816

EVQLLESGGGLVQPGGSLRLSCVASGFTFSTYAMTWVRQAPGKGLEWVS VIRASGDSEIYADSVRGRFTISRDNSKNTVFLQMDSLRVEDTAVYFCANIGQ

RRYCSGDHCYGHFDYWGQGTLVTVSS

Clone No. 817:

QVQLVESGGGVVQPGRSLRLSCAASGFGFNTHGMHWVRQAPGKGLEWL SIISLDGIKTHYADSVKGRFTISRDNSKNTVFLQLSGLRPEDTAVYYCAKDHI GGTNAYFEWTVPFDGWGQGTLVTVSS No. 8

QVTLRESGPAWKPTETLTLTCAFSGFSLNAGRVGVSWIRQPPGQAPEWL ARIDWDDDKAFRTSLKTRLSISKDSSKNQWLTLSNMDPADTATYYCARTQ VFASGGYYLYYLDHWGQGTLVTVSS Clone No. 8

QVQLQESGPGLVKPSQTLSLTCTVSSGAISGADYYWSWIRQPPGKGLEWV GFIYDSGSTYYNPSLRSRVTISIDTSKKQFSLKLTSVTAADTAVYYCARDLGY GGNSYSHSYYYGLDVWGRGTTVTVSS Clone No. 8

QVQLQESGPGLVKPSETLSLTCTVSGGSIGNYYWGWIRQPPGKGLEWIGHI YFGGNTNYNPSLQSRVTISVDTSRNQFSLKLNSVTAADTAVYYCARDSSN WPAGYEDWGQGTLVTVSS Clone No. 825:

QVQLVQSGAEVKKPGASVKVSCKVSGYTFTSNGLSWVRQAPGQGFEWLG WISASSGNKKYAPKFQGRVTLTTDISTSTAYMELRSLRSDDTAVYYCAKDG GTYVPYS DAFDFWGQGTMVTVone No. 827: Clone

QVQLVQSGAEVKKPGASVKVSCRVSGHTFTALSKHWMRQGPGGGLEWM GFFDPEDGDTGYAQKFQGRVTMTEDTATGTAYMELSSLTSDDTAVYYCAT VAAAGN FDNWGQGTLVTVSS Clone No. 829:

QVTLKESGPALVKATQTLTLTCTFSGFSLSRNRMSVSWIRQPPGKALEWLA RIDWDDDKFYNTSLQTRLTISKDTSKNQWLTMTNMDPVDTATYYCARTGI YDSSGYYLYYFDYWGQGTLVTVSS No. 8:

QVQLVQSGAEVKVPGASVKVSCKASGYTFTTYGVSWVRQAPGQGLEWM GWISAYNGNTYYLQKLQGRVTMTTDTSTSTAYMELRGLRSDDTAMYYCAR

DRVGGSSSEVLSRAKNYGLDVWGQGTTVTVSS

Clone No. 831:

QVQLVQSGAEVKKPGASVKVSCKASANIFTYAMHWVRQAPGQRLEWMG WINVGNGQTKYSQRFQGRVTITRDTSATTAYMELSTLRSEDTAVYYCARRA SQYGEVYGNYFDYWGQGTLVTVSS Clone No. 8

QVQLVQSGAEVKRPGASVKVSCKASGYTFISYGFSWVRQAPGQGLEWMG WSSVYNGDTNYAQKFHGRVNMTTDTSTNTAYMELRGLRSDDTAVYFCAR DRNVVLLPAAPFGGMDVWGQGTM38TV

QVQLVESGGGWQPGTSLRLSCAASGFTFSTFGMHWVRQAPGKGLEWVA VISYDGNKKYYADSVKGRFTISRDNSKNTLYLQVNSLRVEDTAVYYCAAQT PYFNESSGLVPDWGQGTLVTVSS Clone No. 841

QVQLVQSGAEVKKPGASVKVSCKASGYTFISFGISWVRQAPGQGLEWMG WISAYNGNTDYAQRLQDRVTMTRDTATSTAYLELRSLKSDDTAVYYCTRD ESMLRGVTEGFGPIDYWGQGTLVTVSS Clone No. 85

EVQLVQSGAEVKKPGQSLKISCKTSGYIFTNYWIGWVRQRPGKGLEWMGV IFPADSDARYSPSFQGQVTISADKSIGTAYLQWSSLKASDTAIYYCARPKYY FDSSGQFSEMYYFDFWGQGTLVTVSS Clone No. 8

QVQLVQSGPEVKKPGASVKVSCKASGYVLTNYAFSWVRQAPGQGLEWLG WISGSNGNTYYAEKFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYFCARD LLRSTYFDYWGQGTLVTVSS Clone No. 8

QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGFSWVRQAPGRGLEWM GWISAYNGNTYYAQNLQGRVTMTTDTSTTTAYMVLRSLRSDDTAMYYCAR DGNTAGVDMWSRDGFDIWGQGTMVTV No. 85

EVQLLESGGGLVQPGGPLRLSCVASGFSFSSYAMNWIRLAPGKGLEWVSG ISGSGGSTYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEP

WIDIWASVISPYYYDGMDVWGQGTTVTVSS

Clone No. 858:

QVQLVQSGAEVKKPGSSVKVSCKASGGSFDGYTISWLRQAPGQGLEWMG RWPTLGFPNYAQKFQGRVTVTADRSTNTAYLELSRLTSEDTAVYYCARM NLGSHSGRPGFDMWGQGTLVTVSS Clone No. 859:

QVQLVESGGGWQPGRSLRLSCAVSGSSFSKYGIHWVRQAPGKGLEWVA VISYDGSKKYFTDSVKGRFTIARDNSQNTVFLQMNSLRAEDTAVYYCATGG GVNVTSWSDVEHSSSLGYWGLGTLVTVSS No. 8

QVQLVESGGGWQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWV AFIWNDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCVKD EVYDSSGYYLYYFDSWGQGTLVTVone No. 8

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVS SISASTVLTYYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCAKDYD FWSGYPGGQYWFFDLWGRGTLVTVSS No. 8

QVQLQESGPGLVTPSETLSVTCTVSNYSIDNAYYWGWIRQPPGKGLEWIG SIHHSGSAYYNSSLKSRATISIDTSKNQFSLNLRSVTAADTAVYYCARDTILT FGEPHWFDPWGQGTLVTVSS Clone No. 870:

QVQLQESGPGLVKPSETLSLTCTVSGDSISNYYWSWIRQPPGKGLEWIGEI SNTWSTNYNPSLKSRVTISLDMPKNQLSLKLSSVTAADTAVYYCARGLFYD SGGYYLFYFQHWGQGTLVTVSS Clone No. 871:

QVQLVESGGGVVQPGRSLRVSCAASGFTFSNYGMHWVRQAPGKGLEWV AVIWYDDSNKQYGDSVKGRFTISRDNSKSTLYLQMDRLRVEDTAVYYCAR ASEYSISWRHRGVLDYWGQGTLVTVSS No. 8

QITLKESGPTLVRPTQTLTLTCTFSGFSLSTSKLGVGWIRQPPGKALEWLAL VDWDDDRRYRPSLKSRLTVTKDTSKNQWLTMTNMDPVDTATYYCAHSAY

YTSSGYYLQYFHHWGPGTLVTVSS

Clone No. 881:

EVQLVESGGGVVQPGGSLRLSCEVSGFTFNSYEMTWVRQAPGKGLEWVS HIGNSGSMIYYADSVKGRFTISRDNAKNSLYLQMNSLRVEDTAVYYCARSD YYDSSGYYLLYLDSWGHGTLV84SS Clone No. 8

QVQLVQSGAEVRKPGASVKVSCKASGHTFINFAMHWVRQAPGQGLEWM GYINAVNGNTQYSQKFQGRVTFTRDTSANTAYMELSSLRSEDTAVYYCAR NNGGSAIIFYYWGQGTLVTVSS Clone No. 886

QVQLVESGGGWQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGLEWV AVISNDGSNKYYADSVKGRFTISRDNSKKTMYLQMNSLRAEDTAVYFCAKT TDQRLLVDWFDPWGQGTLVTVSS Clone No. 888

QLQLQESGPGLVKPSETLSLTCTASGGSINSSNFYWGWIRQPPGKGLEWI GSIFYSGTTYYNPSLKSRVTISVDTSKNQFSLKLSPVTAADTAVYHCARHGF RYCN NGVCSINLDAFDIWGQGTMV94one Clone: 8

QVQLVESGGGWQPGKSLRLSCAASGFRFSDYGMHWVRQAPSKGLEWV AVIWHDGSNIRYADSVRGRFSISRDNSKNTLYLQMNSMRADDTAFYYCAR VPFQIWSGLYFDHWGQGTLVTVSS

These amino acid sequences are in clones encoded by the following nucleic acid sequences, which are also set forth in SEQ ID NOs: 45-88:

Clone No. 735:

caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacgtgcact gtgtctaatggcgccatcggcgactacgactggagctggattcgtcagtccccagggaagggactggagt ggattgggaacataaattacagagggaacaccaactacaacccctccctcaagagtcgagtcaccatgt 30 ccctacgcacgtccacgatgcagttctccctgaagctgagctctgcgaccgctgcggacacggccgtctat tactgtgcgagagatgtaggctacggtggcgggcagtatttcgcgatggacgtctggagcccagggacca cggtcaccgtctcgagt Clone No. 736:

caggtgcagctggtggagtctgggggaggcgtggtccagcctggggggtccctgagactctcctgtacag cgtctggattcaccttcagtacctatggcatgcactgggtccgccaggctcccggcaaggggctggaatgg gtggcatttatacggtatgatggaagtactcaagactatgtagactccgtgaagggccgattcaccatctcc 5 agagacaattccaagaatatggtgtatgtgcagatgaacagcctgagagttgaggacacggctgtctatta ctgtgcgaaagacatggattactatggttcgcggagttattctgtcacctactactacggaatggacgtctgg ggccaagggaccacggtcaccgtctcgagt Clone No. 744:

caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaag 10 gcttctggatacaccttcagcggctattatatgcactgggtgcgacaggcccctggacaagggcttgagtg gatgggatggatcaacactagcagtggtggcacaaactatgcgcagaagtttcagggcagggtcaccat gaccagggacacgtccatcagcacagcccacatggaactgaggaggctgagatctgacgacacggcc gtgtattattgtgcgagagaggacggcaccatgggtactaatagttggtatggctggttcgacccctggggc cagggaaccctggtcaccgtctcgagt 15 Clone No. 793:

caggtgcagctggtggagtctgggggaggcttggtcaagcctggggggtccctgagactctcctgtgcgg cctctggattccccttcggtgactactacatgagctggatccgccaggctccagggaagggactggagtgg gttgcatacattaatagaggtggcactaccatatactacgcagactctgtgaagggccgattcaccatctcc agggacaacgccaagaactccctgtttctgcaaatgaacagcctgagagccggggacacggccctctat 20 tactgtgcgagagggctaattctagcactaccgactgctacggttgagttaggagcttttgatatctggggcc aagggacaatggtcaccgtctcgagt Clone No. 795:

caggtgcagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactg tctctggtgcctccatcagcagtggtgattattactggagttggatccgtcagtctccaaggaagggcctgga 25 gtggattgggtacatcttccacagtgggaccacgtactacaacccgtccctcaagagtcgagctgtcatctc actggacacgtccaagaaccaattctccctgaggctgacgtctgtgactgccgcagacacggccgtctatt attgtgccagagatgtcgacgattttcccgtttggggtatgaatcgatatcttgccctctggggccggggaac cctggtcaccgtctcgagt Clone No. 796:

caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcag cctctggattcagcttcagtcactttggcatgcactgggtccgccaggttccaggcaaggggctggagtggg tggcaattatatcatatgatgggaataatgtacactatgccgactccgtaaagggccgattcaccatctcca gagacaattccaagaacacgctgtttctgcaaatgaacagcctgagagatgacgacacgggtgtgtatta ctgtgcgaaggacgacgtggcgacagatttggctgcctactactacttcgatgtctggggccgtggcaccct

ggtcaccgtctcgagt

Clone No. 799:

5 caggtgcagctggtggagtctgggggcggcgtggtccagcctgggaggtccctgaaactctcttgtgaag cctctggattcaacttcaataattatggcatgcactgggtccgccaggcaccaggcaaggggctggagtg ggtggcagttatttcatatgacggaagaaataagtattttgctgactccgtgaagggccgattcatcatctcca gagacgattccaggaacacagtgtttctgcaaatgaacagcctgcgagttgaagatacggccgtctattac tgtgcgagaggcagcgtacaagtctggctacatttgggactttttgacaactggggccagggaaccctggt caccgtctcgagt 10 Clone No. 800:

caggtgcagctggtggagtctgggggagccgtggtccagcctgggaggtccctgagactctcctgtgaag tgtctggattcagtttcagtgactatggcatgaactgggtccgccagggtccaggcaaggggctggagtgg gtggcagttatatggcatgacggaagtaataaaaattatctagactccgtgaagggccgattcaccgtctcc agagacaattccaagaacacattgtttctgcaaatgaacagcctgagagccgaagacacggctgtatatt actgtgcgaggacgccttacgagttttggagtggctattactttgacttctggggccagggaaccctggtcac 15 cgtctcgagt Clone No. 801:

caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcag cgtctggattccccttcaatagctatgccatgcactgggtccgccaggctccaggcaaggggctggagtgg 20 gtggcagtgatatattatgaagggagtaatgaatattatgcagactccgtgaagggccgattcaccatctcc agagacaattccaagaacactctgtatttgcaaatggatagcctgagagccgaggacacggctgtctatta ctgtgcgaggaagtggctggggatggacttctggggccagggaaccctggtcaccgtctcgagt Clone No. 804:

gaggtgcagctggtggagtctgggggaggcttggtccggcctggggggtccctgagactctcctgttcagc ctctggattcaccttcagtaactatgctatgcactgggtccgccaggctccagggaagagactggaatatgt ttcagctactagtactgatggggggagcacatactacgcagactccctaaagggcacattcaccatctcca gagacaattccaagaacacactgtatcttcaaatgagcagtctcagtactgaggacacggctatttattact gcgcccgccgattctggggatttggaaacttttttgactactggggccggggaaccctggtcaccgtctcga

gt

Clone No. 810:

caggtgcagctggtgcagtctggggctgaggtgaagaagtccgggtcctcggtgaaggtctcctgcaggg

cttctggaggcaccttcggcaattatgctatcaactgggtgcgacaggcccctggacaagggcttgagtgg

gtgggaaggatcatccctgtctttgatacaacaaactacgcacagaagttccagggcagagtcacgatta ccgcggacagatccacaaacacagccatcatgcaactgagcagtctgcgacctcaggacacggccatg tattattgtttgagaggttccacccgtggctgggatactgatggttttgatatctggggccaagggacaatggt caccgtctcgagt Clone No. 811:

caggttcagctggtgcagtctggggctgtcgtggagacgcctggggcctcagtgaaggtctcctgcaagg catctggatacatcttcggcaactactatatccactgggtgcggcaggcccctggacaagggcttgagtgg atggcagttatcaatcccaatggtggtagcacaacttccgcacagaagttccaagacagaatcaccgtga ccagggacacgtccacgaccactgtctatttggaggttgacaacctgagatctgaggacacggccacata ttattgtgcgagacagagatctgtaacagggggctttgacgcgtggcttttaatcccagatgcttctaatacct 10 ggggccaggggacaatggtcaccgtctcgagt Clone No. 812:

caggtgcagctggtgcagtctggggctgagatgaagaagcctgggtcctcggtgaaggtctcctgcaagg cttctggaggctccttcagcagctattctatcagctgggtgcgacaggcccctggacgagggcttgagtggg tgggaatgatcctgcctatctctggtacaacaaactacgcacagacatttcagggcagagtcatcattagc 15 gcggacacatccacgagcacagcctacatggagctgaccagcctcacatctgaagacacggccgtgta tttctgtgcgagagtctttagagaatttagcacctcgacccttgacccctactactttgactactggggccagg gaaccctggtcaccgtctcgagt Clone No. 814:

20 caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaagtccgtgagactctcctgtgtag gctctggcttcaggctcatggactatgctatgcactgggtccgccaggctccaggcaagggactggattgg gtggcagttatttcatatgatggagccaatgaatactacgcagagtccgtgaagggccgattcaccgtctcc agagacaattcagacaacactctgtatctacaaatgaagagcctgagagctgaggacacggctgtgtattt ctgtgcgagagcgggccgttcctctatgaatgaagaagttattatgtactttgacaactggggcctgggaac cctggtcaccgtctcgagt 25 Clone No. 816:

gaggtgcagctgttggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgtagc ctccggattcacctttagtacctacgccatgacctgggtccgccaggctccagggaaggggctggagtgg gtctcagtcattcgtgctagtggtgatagtgaaatctacgcagactccgtgaggggccggttcaccatctcca gagacaattccaagaacacggtgtttctgcaaatggacagcctgagagtcgaggacacggccgtatattt 30 ctgtgcgaatataggccagcgtcggtattgtagtggtgatcactgctacggacactttgactactggggcca gggaaccctggtcaccgtctcgagt Clone No. 817:

csggtgcagctggtggagtctgggggaggcgtggtccaacctgggaggtccctgagactctcctgtgcag cctctggattcggcttcaacacccatggcatgcactgggtccgccaggctccaggcaaggggctggagtg gctgtcaattatctcacttgatgggattaagacccactatgcagactccgtgaagggccgattcaccatctcc agagacaattccaagaacacggtgtttctacaattgagtggcctgagacctgaagacacggctgtatatta ctgtgcgaaagatcatattggggggacgaacgcatattttgaatggacagtcccgtttgacggctggggcc 5 agggaaccctggtcaccgtctcgagt Clone No. 818:

caggtcaccttgagggagtctggtccagcggtggtgaagcccacagaaacgctcactctgacctgcgcct tctctgggttctcactcaacgccggtagagtgggtgtgagttggatccgtcagcccccagggcaggccccg gaatggcttgcacgcattgattgggatgatgataaagcgttccgcacatctctgaagaccagactcagcat 10 ctccaaggactcctccaaaaaccaggtggtccttacactgagcaacatggaccctgcggacacagccac atattactgtgcccggacacaggtcttcgcaagtggaggctactacttgtactaccttgaccactggggcca gggaaccctggtcaccgtctcgagt Clone No. 819:

caggtgcagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactg 15 tctctagtggcgccatcagtggtgctgattactactggagttggatccgccagcccccagggaagggcctg gagtgggttgggttcatctatgacagtgggagcacctactacaacccgtccctcaggagtcgagtgaccat atcaatagacacgtccaagaagcagttctccctgaagctgacctctgtgactgccgcagacacggccgtg tattactgtgccagagatctaggctacggtggtaactcttactcccactcctactactacggtttggacgtctgg ggccgagggaccacggtcaccgtctcgagt 20 Clone No. 824:

caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcact gtctctggtggctccatcggaaattactactggggctggatccggcagcccccagggaagggacttgagt ggattgggcatatctacttcggtggcaacaccaactacaacccttccctccagagtcgagtcaccatttcag tcgacacgtccaggaaccagttctccctgaagttgaactctgtgaccgccgcggacacggccgtgtattac 25 tgtgcgagggatagcagcaactggcccgcaggctatgaggactggggccagggaaccctggtcaccgt ctcgagt

Clone No. 825:

caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaagg tttctggttacacctttaccagtaatggtctcagctgggtgcgacaggcccctggacaagggtttgagtggct 30 gggatggatcagcgctagtagtggaaacaaaaagtatgccccgaaattccagggaagagtcaccttgac cacagacatttccacgagcacagcctacatggaactgaggagtctgagatctgacgatacggccgtatat tactgtgcgaaagatgggggcacctacgtgccctattctgatgcctttgatttctggggccaggggacaatg gtcaccgtctcgagt Clone No. 827:

caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcagg gtttccggacacactttcactgcattatccaaacactggatgcgacagggtcctggaggagggcttgagtg gatgggattttttgatcctgaagatggtgacacaggctacgcacagaagttccagggcagagtcaccatga 5 ccgaggacacagccacaggcacagcctacatggagctgagcagcctgacatctgacgacacggccgt atattattgtgcaacagtagcggcagctggaaactttgacaactggggccagggaaccctggtcaccgtct cgagt

Clone No. 829:

caggtcaccttgaaggagtctggtcctgcgctggtgaaagccacacagaccctgacactgacctgcacct 10 tctctgggttttcactcagtaggaatagaatgagtgtgagctggatccgtcagcccccagggaaggccctg gagtggcttgcacgcattgattgggatgatgataaattctacaacacatctctgcagaccaggctcaccatc tccaaggacacctccaaaaaccaggtggtccttacaatgaccaacatggaccctgtggacacagccacc tattactgcgcacggactgggatatatgatagtagtggttattacctctactactttgactactggggccaggg aaccctggtcaccgtctcgagt 15 Clone No. 830:

caggtgcagctggtgcagtctggagctgaggtgaaggtgcctggggcctcagtgaaggtctcctgcaagg cttctggttacacctttaccacttacggtgtcagctgggtgcggcaggcccctggacaagggcttgagtggat gggttggatcagcgcttacaatggtaacacatactatctacagaagctccagggcagagtcaccatgacc acagacacatccacgagcacagcctacatggagctgcggggcctgaggtctgacgacacggccatgta 20 ttactgtgcgagagatcgtgttgggggcagctcgtccgaggttctatcgcgggccaaaaactacggtttgga cgtctggggccaagggaccacggtcaccgtctcgagt Clone No. 831:

caggttcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagttaaggtttcctgcaaggc ttctgcaaacatcttcacttatgcaatgcattgggtgcgccaggcccccggacaaaggcttgagtggatgg 25 gatggatcaacgttggcaatggtcagacaaaatattcacagaggttccagggcagagtcaccattaccag ggacacgtccgcgactacagcctacatggagctgagcaccctgagatctgaggacacggctgtgtattac tgtgcgaggcgtgcgagccaatatggggaggtctatggcaactactttgactactggggccagggaaccc tggtcaccgtctcgagt Clone No. 835:

caggtgcagctggtgcagtctggagctgaggtgaagaggcctggggcctcagtgaaggtctcctgcaag gcttcaggttacacctttatcagctatggtttcagctgggtgcgacaggcccctggacaagggcttgagtgg atgggatggagcagcgtttacaatggtgacacaaactatgcacagaagttccacggcagagtcaacatg acgactgacacatcgacgaacacggcctacatggaactcaggggcctgagatctgacgacacggccgt gtatttctgtgcgagggatcgcaatgttgttctacttccagctgctccttttggaggtatggacgtctggggcca

agggacaatggtcaccgtctcgagt

Clone No. 838:

5 caggtgcagctggtggagtctgggggaggcgtggtccagccggggacttccctgagactctcctgtgcag cctctggattcaccttcagtacgtttggcatgcactgggtccgccaggctccaggcaaggggctggagtgg gtggcagttatatcatatgatggaaataagaaatactatgcagactccgtgaagggccgattcaccatctcc agagacaattccaagaacacgctgtatctgcaagtgaacagcctgagagtcgaggacacggctgtgtatt actgtgcggcccaaactccatatttcaatgagagcagtgggttagtgccggactggggccagggcaccct ggtcaccgtctcgagt 10 Clone No. 841:

caggtgcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaag gcttctggttacacctttatcagttttggcatcagctgggtgcgacaggcccctggacaaggacttgagtgga tgggatggatcagcgcttacaatggtaacacagactatgcacagaggctccaggacagagtcaccatga ctagagacacagccacgagcacagcctacttggagctgaggagcctgaaatctgacgacacggccgtg 15 tactattgcactagagacgagtcgatgcttcggggagttactgaaggattcggacccattgactactggggc cagggaaccctggtcaccgtctcgagt Clone No. 853:

gaagtgcagctggtgcagtctggagcagaggtgaaaaagccggggcagtctctgaagatctcctgtaag acttctggatacatctttaccaactactggatcggctgggtgcgccagaggcccgggaaaggcctggagt 20 ggatgggggtcatctttcctgctgactctgatgccagatacagcccgtcgttccaaggccaggtcaccatct cagccgacaagtccatcggtactgcctacctgcagtggagtagcctgaaggcctcggacaccgccatat attactgtgcgagaccgaaatattactttgatagtagtgggcaattctccgagatgtactactttgacttctggg gccagggaaccctggtcaccgtctcgagt Clone No. 855:

caggttcagctggtgcagtctggacctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaagg cttctggttatgtgttgaccaactatgccttcagctgggtgcggcaggcccctggacaagggcttgagtggct gggatggatcagcggctccaatggtaacacatactatgcagagaagttccagggccgagtcaccatgac cacagacacatccacgagcacagcctacatggagctgaggagtctgagatctgacgacacggccgttta gt tttctgtgcgagagatcttctgcggtccacttactttgactactggggccagggaaccctggtcaccgtctcga 30

Clone No. 856:

caggtgcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaag

gcttctggttacaccíttíccaactacggtttcagcígggtgcgacaggcccctggacgagggcttgagtgga tgggatggatcagcgcttacaatggtaacacatactatgcacagaacctccagggcagagtcaccatga ccacagacacatccacgaccacagcctacatggtactgaggagcctgagatctgacgacacggccatg tattactgtgcgagagatggaaatacagcaggggttgatatgtggtcgcgtgatggttttgatatctggggcc aggggacaatggtcaccgtctcgagt Clone No. 857:

gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggcccctgaggctctcctgtgtagc ctctggattcagctttagcagctatgccatgaactggatccgcctggctccagggaaggggctggagtggg tctcaggtattagtggtagcggtggtagcacttactacggagactccgtgaagggccggttcaccatctcca gagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatatt 10 actgtgegaaagagecgtggatcgatatagtagtggcatetgttatatccccctactactacgacggaatgg acgtctggggccaagggaccacggtcaccgtctcgagt Clone No. 858:

caggttcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagg cctctggaggatccttcgacggctacactatcagctggctgcgacaggcccctggacaggggcttgagtg 15 gatgggaagggtcgtccctacacttggttttccaaactacgcacagaagttccaaggcagagtcaccgtta ccgcggacagatccaccaacacagcctacttggaattgagcagactgacatctgaagacacggccgtat attactgtgcgaggatgaatctcggatcgcatagcgggcgccccgggttcgacatgtggggccaaggaa ccctggtcaccgtctcgagt Clone No. 859:

caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccttgagactctcctgtgcagt gtctggatccagcttcagtaaatatggcatacactgggtccgccaggctccaggcaaggggctggagtgg gtggcagttatatcgtatgatggaagtaaaaagtatttcacagactccgtgaagggccgattcaccatcgcc agagacaattcccagaacacggtttttctgcaaatgaacagcctgagagccgaggacacggctgtctatt actgtgcgacaggagggggtgttaatgtcacctcgtggtccgacgtagagcactcgtcgtccttaggctact 25 ggggcctgggaaccctggtcaccgtctcgagt Clone No. 861:

caggtgcagctggtggagtctgggggaggcgtggtccagcctggggggtccctgagactctcctgtgcag cgtctggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgg gtggcatttatatggaatgatggaagtaataaatactatgcagactccgtgaagggccgattcaccatctcc 30 agagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagctgaggacacggctgtgtatt actgtgtgaaagatgaggtctatgatagtagtggttattacctgtactactttgactcttggggccagggaacc ctggtcaccgtctcgagt Clone No. 863:

gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagc ctctggattcacgtttagctcctataccatgagctgggtccgccaggctccagggaaggggctggagtggg tctcaagtattagtgctagtactgttctcacatactacgcagactccgtgaagggccgcttcaccatctccag 5 agacaattccaagaacacgctgtatctgcaaatgagtagcctgagagccgaggacacggccgtatatta ctgtgcgaaagattacgatttttggagtggctatcccgggggacagtactggttcttcgatctctggggccgtg gcaccctggtcaccgtctcgagt Clone No. 868:

caggtgcagctgcaggagtcgggcccaggactggtgacgccttcggagaccctgtccgtcacttgcactg 10 tctctaattattccatcgacaatgcttactactggggctggatccggcagcccccagggaagggtctggagt ggataggcagtatccatcatagtgggagcgcctactacaattcgtccctcaagagtcgagccaccatatct atagacacgtccaagaaccaattctcgttgaacctgaggtctgtgaccgccgcagacacggccgtatatta ctgtgcgcgcgataccatcctcacgttcggggagccccactggttcgacccctggggccagggaaccctg gtcaccgtctcgagt 15 Clone No. 870:

caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccttgtccctcacctgcactg tctcaggtgactccatcagtaattactactggagttggatccggcagcccccagggaagggactggagtg gattggagaaatatctaacacttggagcaccaattacaacccctccctcaagagtcgagtcaccatatctct agacatgcccaagaaccagttgtccctgaagctgagctctgtgaccgctgcggacacggccgtatattact 20 gtgcgagagggcttttctatgacagtggtggttactacttgttttacttccaacactggggccagggcaccctg gtcaccgtctcgagt Clone No. 871:

caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagagtctcctgtgcag cgtctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgg 25 gtggcagttatatggtatgatgacagtaataaacagtatggagactccgtgaagggccgattcaccatctcc agagacaattccaagagtacgctgtatctgcaaatggacagactgagagtcgaggacacggctgtgtatt attgtgcgagagcctccgagtatagtatcagctggcgacacaggggggtccttgactactggggccaggg aaccctggtcaccgtctcgagt Clone No. 880:

cagatcaccttgaaggagtctggtcctacgctggtgagacccacacagaccctcacactgacctgcacctt ctctgggttctcactcagcactagtaaactgggtgtgggctggatccgtcagcccccaggaaaggccctgg agtggcttgcactcgttgattgggatgatgataggcgctacaggccatctttgaagagcaggctcaccgtca ccaaggacacctccaaaaaccaggtggtccttacaatgaccaacatggaccctgtggacacagccaca tattactgtgcacacagtgcctactatactagtagtggttattaccttcaatacttccatcactggggcccgggc

accctggtcaccgtctcgagt

Clone No. 881:

5 gaggtgcagctggtggagtctgggggaggcgtggtacagcctggaggctccctgagactctcctgtgaag tctccggattcaccttcaatagttatgaaatgacctgggtccgccaggccccagggaaggggctggagtg ggtttcacacattggtaatagtggttctatgatatactacgctgactctgtgaagggccgattcaccatctcca gagacaacgccaagaactcactatatctgcaaatgaacagcctgagagtcgaggacacggctgtttatta ctgtgcgaggtcagattactatgatagtagtggttattatctcctctacttagactcctggggccatggaaccct ggtcaccgtctcgagt 10 Clone No. 884:

caggtgcagctggtgcagtctggggctgaggtgaggaagcctggggcctcagtgaaggtttcctgcaagg cttctggacatactttcattaactttgctatgcattgggtgcgccaggcccccggacaggggcttgagtggat gggatacatcaacgctgtcaatggtaacacacagtattcacagaagttccagggcagagtcacctttacg agggacacatccgcgaacacagcctacatggagctgagcagcctgagatctgaagacacggctgtgta 15 ttactgtgcgagaaacaatgggggctctgctatcattttttactactggggccagggaaccctggtcaccgtc tcgagt

Clone No. 886:

caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcag cctctggattcagcttcagtagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgg 20 gtggcagttatatcaaatgatggaagtaataaatactatgcagactccgtgaagggccgattcaccatctcc agagacaattccaagaaaacgatgtatctgcaaatgaacagcctgagagctgaggacacggctgtgtat ttctgtgcgaagacaacagaccagcggctattagtggactggttcgacccctggggccagggaaccctgg tcaccgtctcgagt Clone No. 888:

cagctgcagctgcaggagtcgggcccaggactggtgaagccatcggagaccctgtccctcacctgcact gcctctggtggctccatcaacagtagtaatttctactggggctggatccgccagcccccagggaaggggct ggagtggattgggagtatcttttatagtgggaccacctactacaacccgtccctcaagagtcgagtcaccat atccgtagacacgtccaagaaccagttctccctgaagctgagccctgtgaccgccgcagacacggctgtc tatcactgtgcgagacatggcttccggtattgtaataatggtgtatgctctataaatctcgatgcttttgatatctg 30 gggccaagggacaatggtcaccgtctcgagt Clone No.894:

caggtgcagctggtggagtctgggggaggcgtcgtccagcctggaaagtccctgagactctcctgtgcag

cgtctggaítagagícícactacggcatgcactgggtccggcaggctccaagcaaggggctggagtg ggtggcagttatctggcatgacggaagtaatataaggtatgcagactccgtgaggggccgattttccatctc

cagagacaattccaagaacacgctgtatttgcaaatgaacagcatgagagccgacgacacggctttttatt

attgtgcgagagtcccgttccagatttggagtggtctttattttgaccactggggccagggaaccctggtcac

cgtctcgagt

In the same clones, complete amino acid sequences

Light chains (ie light chains that include constant and variable regions) have the following amino acid sequences, which are also set forth in SEQ ID NOs: 89-132:

Clone No. 735:

EIVLTQSPATLSLSPGERATLSCRASQSVNSHLAWYQQKPGQAPRLLIYNT FNRVTGIPARFSGSGSGTDFTLTISSLATEDFGVYYCQQRSNWPPALTFGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 15 Clone No. 736:

DIQMTQSPSSLSASVGDRVTFTCRASQRISNHLNWYQQKPGKAPKLLIFGA STLQSGAPSRFSGSGSGTDFTLTITNVQPDDFATYYCQQSYRTPPINFGQG TRLDIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS 20 PVTKSFNRGEC Clone No. 744:

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYG ASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDSSLSTWTFG QGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV 25 DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC Clone No. 793:

DIQMTQSPSSLSASVGDRVTITCRASQSITGYLNWYQQKPGKAPKLLIYATS TLQSEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC Clone No. 795:

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIHG ASTGATGTPDRFSGSGSGTDFTLTISTLEPEDFAVYYCQQYGRTPYTFGQG TKLENKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDN 5 ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC Clone No. 796:

DIVMTQTPLSLSVTPGQPASISCRSSQSLLRSDGKTFLYWYLQKPGQSPQP LMYEVSSRFSGVPDRFSGSGSGADFTLNISRVETEDVGIYYCMQGLKIRRT 10 FGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC Clone No. 799:

15 DIQMTQSPSTLSASVGDRVTFSCRASQSVSSWVAWYQQKPGKAPKLLISE ASNLESGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYHSYSGYTFGQ GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC Clone No. 800:

AIQLTQSPSSLSASVGDRVTLTCRASQGITDSLAWYQQKPGKAPKVLLYAA SRLESGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYSKSPATFGPGT KVEIRRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKS F 25 N RG EC Clone No. 801:

DIVMTQSPLSLPVTPGEPASISCRSSQSLLNSNGFNYVDWYLQKPGQSPQL LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALETPLT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH 30 QGLSSPVTKSFNRGEC Clone No. 804:

EIVLTQSPGTLSLSPGGRATLSCRASQSVSSGYLAWYQQKPGQAPRLLIYG

ASGRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYFGSPYTFGQG TKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC Clone No. 810:

NIQMTQSPSAMSASVGDRVTITCRASQGISNYLVWFQQKPGKVPKRLIYAA SSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNISPYTFGQGT KLETKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 10 Clone No. 811:

DIVMTQSPDSLAVSLGERATINCRSSETVLYTSKNQSYLAWYQQKARQPPK LLLYW ASTRESGVPARFSGSGSGTDFTLAISSLQAEDVAVYYCQQFFRSPF TFGPGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTH 15 QGLSSPVTKSFNRGEC Clone No. 812:

EIVLTQSPGTLSLSPGERVTLSCRASQSVSSSYIAWYQQKPGQAPRLVIYAA SRRATGVPDRFSGSGSATDFTLTISRLEPEDLAVYYCQHYGNSLFTFGPGT KVDVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA 20 LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC Clone No. 814:

DIQMTQSPSTLSASVGDRVTITCRASQSIGSRLAWYQQQPGKAPKFLIYDA SSLESGVPSRFSGSGSGTEFTLTISSLQPEDLATYYCQQYNRDSPWTFGQ 25 GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC Clone No. 816:

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGRYYVDWYLQKPGQSPHL LIYLASNRASGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCMQGLHTPWT FGQGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPXiTKSFNRGEC Clone No. 817:

EIVMTQSPATLSASPGERATLSCWASQTIGGNLAWYQQKPGQAPRLLIYGA STRATGVPARFSGSGSGTEFTLAISSLQSEDFAVYYCQQYKNWYTFGQGT KLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNA 5 LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC Clone No. 818:

DIQMTQSPSSLSASVGDRVTITCRASQTIASYVNWYQQKPGRAPSLLIYAAS NLQSGVPPRFSGSGSGTDFTLTISGLQPDDFATYYCQQSYSYRALTFGGG 10 TKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC Clone No. 819:

15 EIVLTQSPATLSLSPGERATLSCRASQSVSSSLAWYQQTPGQAPRLLIYDAS YRVTGIPARFSGSGSGIDFTLTISSLEPEDFAVYYCQQRSNWPPGLTFGGG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC Clone No. 824:

AIQLTQSPSSLSASVGDTVTVTCRPSQDISSALAWYQQKPGKPPKLLIYGAS TLDYGVPLRFSGTASGTHFTLTISSLQPEDFATYYCQQFNTYPFTFGPGTKV DIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC 25 Clone No. 825:

DIVMTQSPDSLAVSLGERATINCKSSQSVLYNSNNKNYLAWYQQKPGQPP KLLIHLASTREYGVPDRFSGSGSGTDFALIISSLQAEDVAVYYCQQYYQTPL TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT 30 HQGLSSPVTKSFNRGEC Clone No. 827:

DIQMTQSPSSLAASVGDRVTITCRASQFISSYLHWYQQRPGKAPKLLMYAA

STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTNPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDLSKYLTKKNYVKKKLNSSY

DIQMTQSPSSLSASVGDRVTITCRASQSIASYLNWYQQKPGKAPKLLIYAAS SLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSYSTRFTFGPGTK VDVKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 10 Clone No. 830:

DIQMTQSPSTLSASVGDRVTITCRASQSVTSELAWYQQKPGKAPNFLIYKA SSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSFPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP 15 VTKSFNRGEC Clone No. 831:

DIQMTQSPSTLSASVGDRLTITCRASQNIYNWLAWYQQKPGKAPKLLIYDA STLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSLSPTFGQGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL 20 QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC Clone No. 835:

DIQLTQSPSFLSASLEDRVTITCRASQGISSYLAWYQQKPGKAPKLLLDAAS TLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPRTFGQGTK 25 VDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC Clone No. 838:

DIQMTQSPSSLSASVGDRVSITCRASQGISNYLAWYQQKPGKVPKLLIYAAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPQTFGQGTK VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC Clone No. 841:

DIVMTQSPDSLAVSLGERATINCRSSQSVLYSSNNKNYLAWYQQKPGQPP KLLVYW ASTRASGVPDRFSGSGSGTDFTLTLSSLQAEDVAVYYCQQFHST PRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKV 5 QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC Clone No. 853:

EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYQQKPGQAPRLLIYG ASSRAAGMPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGNSPLTFGG 10 GTEVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNF.YPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC Clone No. 855:

DIQMTQSPSSVSASVGDRVTITCRASQAISNWLAWYQQKPGKAPKLLIYAA 15 SSLQSGVPSRFSGSGSGTDFTLTISGLQPEDFATYYCQQADTFPFTFGPGT KVDIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC Clone No. 856:

DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSNDGNTYLDWYLQKPGQSPQ LLIYTFSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQRIEFPYT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC 25 Clone No. 857:

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHRNEYNYLDWYLQKPGQSPQL LIYWGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQTLQTPR TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT 30 HQGLSSPVTKSFNRGEC Clone No. 858:

DIQMTQSPSSVSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIFDA

TKLETGVPTRFIGSGSGTDFTVTITSLQPEDVATYYCQHFANLPYTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSKLTLSKYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYY HAYYYYYYYYYYY HAYYYYYYYYY have been ...

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKVPKLLVFAA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNSAPLTFGGGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 10 Clone No. 861:

DIQMTQSPSSLSASVGDRVTITCRASQIIASYLNWYQQKPGRAPKLLIYAAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPIFTFGPGTK VNIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP 15 VTKSFNRGEC Clone No. 863:

EIVLTQSPATLSLSPGERATLSCRTSQSVSSYLAWYQQKPGQAPRLLIYDAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQ 20 SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC Clone No. 868:

EIVMTQSPATLSVSPGERATLSCRASQSIKNNLAWYQVKPGQAPRLLTSGA SARATGIPGRFSGSGSGTDFTLTISSLQSEDIAVYYCQEYNNWPLLTFGGG 25 TKVEIQRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC Clone No. 870:

DIQMTQSPPSLSASVGDRVTITCRASQRIASYLNWYQQKPGRAPKLLIFAAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDYATYYCQQSYSTPIYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC Clone No. 871:

DIQMTQSPSSLSASVGDRVTITCQASQGISNYLNWYQQKPGKAPKLLIFDA SNLESEVPSRFSGRGSGTDFTFSISSLQPEDIATYFCQQYDNFPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL 5 QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC Clone No. 880:

DIQMTQSPSSLAASVGDRVTITCRASQTIASYVNWYQQKPGKAPNLLIYAAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQSYSFPYTFGQGTK 10 LDIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC Clone No. 881:

15 DIQMTQSPSSLSASVGDRVTITCRASQTIASYVNWYQQKPGKAPKLLIYAAS NLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSVPRLTFGGGT KVDITRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC Clone No. 884:

DIQMTQSPSSLSASVGDRVTITCRSSQTISVFLNWYQQKPGKAPKLLIYAAS SLHSAVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQESFSSSTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC 25 Clone No. 886:

EIVMTQSPATLSVSPGETATLSCRASQSVSSNLAWYQHKPGQAPRLLIHSA STRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNMWPPWTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL 30 SSPVTKSFNRGEC Clone No. 888:

DIVMTQSPLSLPVTPGAPASISCRSSQSLLRTNGYNYLDWYLQKPGQSPQL

LIYLGSIRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSLQTSITF GQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDLSKYLTKKNYVKKYLVSKYLKY

EIVMTQSPATLSVSPGERATLSCRASQSVGNNLAWYQQRPGQAPRLLIYG ASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYDKWPETFGQG TKVDIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC

The light chain encoding nucleic acid fragments

These clones have the following nucleic acid sequences, which are also provided as SEQ ID NOs: 133-176

Clone No 735:

15 gaaattgtgttgacacagtctccagccaccctgtccttgtctccaggagaaagagccaccctctcctgcagg gccagtcagagtgttaacagccacttagcctggtaccaacagaaacctggccaggctcccaggctcctca tctataatacattcaatagggtcactggcatcccagccaggttcagtggcagtgggtctgggacagacttca ctctcaccatcagcagccttgcgactgaagattttggcgtttattactgtcagcagcgtagcaactggcctcc cgccctcactttcggcggagggaccaaagtggagatcaaacgaactgtggctgcaccatctgtcttcatctt cccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagag 20 aggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagag caggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgag aaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaac aggggagagtgt

Clone No 736:

gacatccagatgacccagtctccatcctccctgtctgcatctgtgggagacagagtcaccttcacttgccgg gccagtcagaggattagcaaccatttaaattggtatcaacaaaagccagggaaagcccctaaactcctg atctttggtgcatccactcttcaaagtggggccccatcaaggttcagtggcagtggatctgggacagatttca ctctcaccatcactaatgtacaacctgacgattttgcaacttactactgtcaacagagttacagaactccccc gatcaacttcggccaagggacacgcctggacattaagcgaactgtggctgcaccatctgtcttcatcttccc 30 gccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagag gccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagca ggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaa acacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacag

gggagagtgt

Clone No 744:

5 gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagg gccagtcagagtgttagcagcagctacttagcctggtatcagcagaaacctggccaggctcccaggctcc tcatctatggtgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacaga cttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtatgatagctcac tttctacgtggacgttcggccaagggaccaaggtggaaatcaaacgaactgtggctgcaccatctgtcttc atcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatccc 10 agagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcac agagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagacta cgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagct tcaacaggggagagtgt

Clone No 793:

15 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgg gcaagtcagagcattaccggctatttaaattggtatcagcagaaaccagggaaagcccctaaactcctga tctatgctacatccactttgcaaagtgaggtcccatcaaggttcagtggcagtggatctgggacagatttcac tctcaccatcagcagtcttcaacctgaagattttgcaacttactactgtcaacagagttataataccctcacttt cggcggagggaccaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctg 20 atgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagt acagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagc aaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaa gtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggaga GTGT

25 Clone No 795:

gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagg gccagtcagagtgttagcagcagctacttagcctggtatcagcagaaacctggccaggctcccaggctcc tcatacatggcgcatccaccggggccactggcaccccagacaggttcagtggcagtgggtctgggacag acttcactctcaccatcagtacactggagcctgaagattttgcagtgtattactgtcagcaatatggtaggac 1 30 accgtacacttttggccaggggaccaagctggagaacaaacgaactgtggctgcaccatctgtcttcatctt cccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagag aggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagag

caggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgag

aaacacaaagtctacgcctgcgaagtcacccatcagggcccctgagctcgcccgtcacaaagagcttcaac

aggggagagtgt

Clone No 796:

gatattgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcctgcaggt ctagtcagagcctcctgcgaagtgatggaaagacgtttttgtattggtatctgcagaagccaggccagtctc cccaacccctaatgtatgaggtgtccagccggttctctggagtgccagataggttcagtggcagcgggtca ggggcagatttcacactgaacatcagccgggtggagactgaggatgttgggatctattactgcatgcaag 10 gtttgaaaattcgtcggacgtttggcccagggaccaaggtcgaaatcaagcgaactgtggctgcaccatct gtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttct atcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagt gtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagca gactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaa 15 gagcttcaacaggggagagtgt

Clone No 799:

gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccttctcttgccggg ccagtcagagtgttagtagttgggtggcctggtatcagcagaaaccaggaaaagcccctaagctcctgat ctctgaggcctccaatttggaaagtggggtcccatcccggttcagcggcagtggatccgggacagaattca 20 ctctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacagtatcatagttactctggg tacacttttggccaggggaccaagttggaaatcaagcgaactgtggctgcaccatctgtcttcatcttcccgc catctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggc caaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcagg acagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaac 25 acaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagg ggagagtgt

Clone No 800:

gccatccagttgacccagtctccatcgtccctgtctgcatctgtaggcgacagagtcaccctcacttgccgg gcgagtcagggcattaccgattctttagcctggtatcagcagaaaccagggaaagcccctaaggtcctgct ctatgctgcttccagattggaaagtggggtcccatccaggttcagtggccgtggatctgggacggatttcact ctcaccatcagcagcctgcagcctgaagactttgcaacttattactgtcaacagtattctaagtcccctgcga cgttcggcccagggaccaaggtggaaatcagacgaactgtggctgcaccatctgtcttcatcttcccgcca tctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcca aagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggac agcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacac 5 aaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggg agagtgt

Clone No 801:

gatattgtgatgacccagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggt ctagtcagagcctcctaaatagtaatggattcaactatgtggattggtacctgcagaagccagggcagtctc 10 cacaactcctgatctatttgggttctaatcgggcctccggggtccctgacaggttcagtggcagtggatcagg cacagattttacactgaaaatcagcagagtggaggctgaggatgttggggtttattactgcatgcaagctct agaaactccgctcactttcggcggagggaccaaggtggagatcaaacgaactgtggctgcaccatctgtc ttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatc ccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtc 15 acagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcaga ctacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaaga gcttcaacaggggagagtgt

Clone No 804:

20 gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccagggggaagagccaccctctcctgcagg gccagtcagagtgttagcagcggctacttagcctggtaccagcagaaacctggccaggctcccaggctc ctcatctatggtgcatccggcagggccactggcatcccagacaggttcagtggcagtgggtctgggacag acttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtattttggctcac cgtacacttttggccaggggaccaagctggagctcaaacgaactgtggctgcaccatctgtcttcatcttcc cgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagaga 25 ggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagc aggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgaga aacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaaca ggggagagtgt

Clone No 810:

aacatccagatgacccagtctccatctgccatgtctgcatctgtaggagacagagtcaccatcacttgtcgg gcgagtcagggcattagtaattatttagtctggtttcagcagaaaccagggaaagtccctaagcgcctgatc tatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcact ctcacaatcagcagcctgcagcctgaagattttgcaacttattactgtctacagcataatatttccccttacact tttggccaggggaccaagctggagaccaaacgaactgtggctgcaccatctgtcttcatcttcccgccatct gatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaa 5 gtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacag caaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaa agtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggag agtgt

Clone No 811:

gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgca ggtccagtgagactgttttatacacctctaaaaatcagagctacttagcttggtaccagcagaaagcacga cagcctcctaaactactcctttactgggcatctacccgggaatccggggtccctgcccgattcagtggcagc ggatctgggacagatttcactctcgccatcagcagcctgcaggctgaagatgtggcagtttattactgtcag caattttttaggagtcctttcactttcggccccgggaccagactggagattaaacgaactgtggctgcaccat 15 ctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataactt ctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggaga gtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaag cagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcaca aagagcttcaacaggggagagtgt

Clone No 812:

gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagttaccctctcttgcaggg ccagtcagagtgttagcagcagttacatagcctggtaccagcagaagcctggccaggctcccaggctcgt catctatgctgcatcccgcagggccactggcgtcccagacaggttcagtggcagtgggtctgcgacagac ttcactctcaccatcagtagactggagcctgaagatcttgcagtgtattactgtcagcactatggtaactcact 25 attcactttcggccctgggaccaaggtggatgtcaaacgaactgtggctgcaccatctgtcttcatcttcccg ccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagagg ccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcag gacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaa cacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagg 30 ggagagtgt Clone No 814:

gacatccagatgacccagtctccctccaccctgtctgcatctgtcggagacagagtcaccatcacttgccg ggccagtcagagtattggtagccggttggcctggtatcagcagcaaccagggaaagcccctaaattcctg atctatgatgcctccagtttggaaagtggggtcccatcaaggttcagcggcagtggatcagggacagaatt 5 cactctcaccatcagcagcctgcagccggaggatcttgcaacttattactgccaacagtacaatagagatt ctccgtggacgttcggccaagggaccaaggtggaaatcaagcgaactgtggctgcaccatctgtcttcatc ttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccaga gaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacaga gcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacga 10 gaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttca acaggggagagtgt

Clone No 816:

gatattgtgatgacccagtctccactctccctgcccgtcaccccaggagagccggcctccatctcctgcagg tctagtcagagcctcctgcatagtgatggacgctactatgtggattggtacctgcagaagccagggcagtct 15 ccacacctcctgatctatttggcttctaatcgggcctccggggtccctgacaggttcactggcagtggatcag gcacagattttacactgaaaatcagcagagtggaggctgaggatgttggcgtttattactgcatgcaaggtc tacacactccttggacgttcggccaggggaccaaggtggacatcaagcgaactgtggctgcaccatctgt cttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctat cccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgt 20 cacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcag actacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaag agcttcaacaggggagagtgt

Clone No 817:

gaaattgtaatgacacagtctccagccaccctgtctgcgtccccaggggaaagagccaccctctcctgttg 25 ggccagtcagactattggaggcaacttagcctggtaccagcagaaacctggccaggctcccaggctcct catctatggtgcatccaccagggccactggtgtcccagccaggttcagtggcagtgggtctgggacagagt tcactctcgccatcagcagcctgcagtctgaagattttgcagtttattactgtcagcagtataaaaactggtac acttttggccaggggaccaagctggagctcaaacgaactgtggctgcaccatctgtcttcatcttcccgcca tctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcca 30 aagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggac agcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacac Λ

aaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggg

agagtgt

Clone No 818:

5 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgg gcaagtcagaccattgccagttacgtaaattggtaccaacaaaaaccagggagagcccctagtctcctga tctatgctgcatctaacttgcagagtggggtcccaccaaggttcagtggcagtggatctgggacagacttca ctctcaccatcagcggtctgcaacctgacgattttgcaacttattactgtcaacagagttacagttatcgagcg ctcactttcggcggagggaccaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccg ccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagagg 10 ccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcag gacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaa cacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagg ggagagtgt

Clone No 819:

gaaattgtgttgacacagtctccagccaccctgtcgttgtccccaggggaaagagccaccctctcctgcag ggccagtcagagtgttagcagctccttagcctggtaccaacagacacctggccaggctcccaggcttctca tctatgatgcgtcctacagggtcactggcatcccagccaggttcagtggcagtgggtctgggatagacttca ctctcaccatcagcagcctagagcctgaagattttgcagtttactattgtcagcagcgtagcaactggcctcc ggggctcactttcggcggggggaccaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatct 20 tcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccaga gaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacaga gcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacga gaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttca acaggggagagtgt

Clone No 824:

gccatccagttgacccagtctccatcctccctgtctgcatctgttggagacacagtcaccgtcacttgccggc caagtcaggacattagcagtgctttagcctggtatcagcagaaaccagggaaacctcctaagctcctgatc tatggtgcctccactttggattatggggtcccattaaggttcagcggcactgcatctgggacacatttcactctc accatcagcagcctgcaacctgaagattttgcaacttattactgtcaacagtttaatacttacccattcactttc 30 ggccctgggaccaaagtggatatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgat gagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagta cagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagca

aggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaag

tctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagt

gt

Clone No 825:

gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgca agtccagccagagtgttttatacaactccaacaataagaactacttagcctggtatcagcagaaaccagg acagcctcctaagctcctcattcacttggcatctacccgggaatacggggtccctgaccgattcagtggcag cgggtctgggacagatttcgctctcatcatcagcagcctgcaggctgaagatgtggcagtttattactgtcaa 10 caatattatcaaactcctctaacttttggccaggggaccaaggtggagatcaaacgaactgtggctgcacc atctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataac ttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggag agtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaa gcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcac 15 aaagagcttcaacaggggagagtgt

Clone No 827:

gacatccagatgacccagtctccatcctccctggctgcatctgtaggagacagagtcaccatcacttgccg. ggcaagtcagttcattagcagctatttacattggtatcagcaaagaccaggcaaggcccctaaactcctga tgtatgctgcctccactttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcac 20 tctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacactaacccatac acttttggccaggggaccaagctggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgcca tctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcca aagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggac agcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacac 25 aaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggg agagtgt

Clone No 829:

gacatccagatgacccagtctccatcctccctatctgcatctgtaggagacagagtcaccatcacttgccgg gcaagtcagagcattgccagctatttaaattggtatcagcagaaaccagggaaagcccccaaactcctg atctatgctgcatccagtttgcatagtggggtcccatcaagattcagtggcagtggatctgggacagatttca ctctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacacagttacagtactcgattc actttcggccctgggaccaaagtggatgtcaaacgaactgtggctgcaccatctgtcttcatcttcccgccat ctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaa agtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggaca gcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacaca 5 aagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga gagtgt

Clone No 830:

gacatccagatgacccagtctccttcgaccctgtctgcatctgtaggagacagagtcaccatcacttgccgg gccagtcagagtgttactagtgagttggcctggtatcagcagaaaccagggaaagcccctaacttcctgat 10 ctataaggcgtctagtttagaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattca ctctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacagtataatagttttccgtaca cttttggccaggggaccaagctggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccat ctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaa agtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggaca 15 gcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacaca aagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga gagtgt

Clone No 831:

20 gacatccagatgacccagtctccttccaccctgtctgcatctgtaggcgacagactcaccatcacttgccgg gccagtcagaatatttataactggttggcctggtatcagcagaaaccagggaaagcccctaaactcctgat ctatgacgcctccactttggaaagtggggtcccatcaaggttcagcggcagtggatctgggacagagttca ctctcaccatcagcagcctgcagcctgatgattttgcgacttattactgccaacaatataatagtttgtctccga cgttcggccaagggaccaaggtggaaatcaagcgaactgtggctgcaccatctgtcttcatcttcccgcca tctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcca 25 aagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggac agcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacac aaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggg agagtgt

Clone No 835:

gacatccagttgacccagtctccatccttcctgtctgcatctttagaagacagagtcactatcacttgccgggc cagtcagggcattagcagttatttagcctggtatcagcaaaaaccagggaaagcccctaagctcctgctcg atgctgcatccactttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagagttcact ctcacaatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagcttaatagttaccctcgga cgttcggccaagggaccaaggtggacatcaaacgaactgtggctgcaccatctgtcttcatcttcccgcca tctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcca 5 aagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggac agcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacac aaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggg agagtgt

Clone No 838:

10 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcagcatcacttgccgg gcgagtcagggcattagcaattatttagcctggtatcagcagaaaccagggaaggttcctaagctcctgat ctatgctgcatccactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggacagatttcactc tcaccatcagcagcctgcagcctgaggatgttgcaacttattactgtcaaaagtataacagtgcccctcaaa cgttcggccaagggaccaaggtggaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgcca 15 tctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcca aagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggac agcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacac aaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggg agagtgt

I 20 Clone No 841:

gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgca ggtccagccagagtgttttatacagctccaacaataagaactacttagcttggtaccagcagaaaccagg acagcctcctaagctgctcgtttactgggcatcaacccgggcatccggggtccctgaccgattcagtggca gcgggtctgggacagatttcactctcaccctcagcagcctgcaggctgaagatgtggcagtttattactgtca 25 gcagtttcatagtactcctcggacgttcggccaagggaccaaggtggagatcaaacgaactgtggctgca ccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaata acttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccagg agagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagca aagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtc 30 acaaagagcttcaacaggggagagtgt Clone No 853:

gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagg gccagtcagagtgttagcagcaactacttagcctggtaccagcagaaacctggccaggctcccaggctc ctcatctatggtgcatccagcagggccgctggcatgccagacaggttcagtggcagtgggtctgggacag 5 acttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtatggtaactc accgctcactttcggcggagggaccgaggtggagatcaaacgaactgtggctgcaccatctgtcttcatctt cccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagag aggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagag caggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgag 10 aaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaac aggggagagtgt

Clone No 855:

gacatccagatgacccagtctccatcttctgtgtctgcatctgtaggagacagagtcaccatcacttgtcggg cgagtcaggctattagtaactggttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgat 15 ctatgctgcatccagtttgcaaagtggggtcccatcaagattcagcggcagtggatctgggacagatttcac tctcactatcagcggcctgcagcctgaggattttgcaacttactattgtcaacaggctgacactttccctttcac tttcggccctgggaccaaagtggatatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatct gatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaa gtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacag 20 caaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaa agtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggag agtgt

Clone No 856:

gatattgtgatgacccagactccactctccctgcccgtcacccctggagagccggcctccatctcctgcagg 25 tctagtcagagcctcttggatagtaatgatggaaacacctatttggactggtacctgcagaagccagggca gtctccacagctcctgatttatacattttcctatcgggcctctggagtcccagacaggttcagtggcagtgggt ctggcactgatttcacactgaaaatcagcagggtggaggccgaggatgttggagtttattactgcatgcaac gtatcgagtttccgtacacttttggccaggggaccaagctggagatcaaacgaactgtggctgcaccatct gtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttct 30 atcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagt gtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagca gactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaa

gagcttcaacaggggagagtgt

Clone No 857:

5 gatattgtgatgacccagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggt ctagtcagagcctcctgcatagaaatgagtacaactatttggattggtacttgcagaagccagggcagtctc cacagctcctgatctattggggttctaatcgggcctccggggtccctgacaggttcagtggcagtggatcag gcacagattttacactgaaaatcagcagagtggaggctgaggatgttggggtttattactgcatgcaaactc tacaaactcctcggacgttcggccaagggaccaaggtggaaatcaaacgaactgtggctgcaccatctgt cttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctat 10 cccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgt cacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcag actacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaag agcttcaacaggggagagtgt

Clone No 858:

gacatccagatgacccagtctccatcctccgtgtctgcatctgtgggagacagagtcaccatcacttgccag gcgagtcaagacattagcaactatttaaattggtatcagcagaaaccagggaaagcccctaagctcctga tcttcgatgcaaccaaattggagacaggggtcccaacaaggttcattggaagtggatctgggacagatttt actgtcaccatcaccagcctgcagcctgaagatgttgcaacatattactgtcaacactttgctaatctcccat acacttttggccaggggaccaagctggagatcaagcgaactgtggctgcaccatctgtcttcatcttcccgc 20 catctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggc caaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcagg acagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaac acaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagg ggagagtgt

Clone No 859:

gacatccagatgacccagtctccatcttccctgtctgcatctgtaggagacagagtcaccatcacttgccgg gcgagtcagggcattaggaattatttagcctggtatcagcagaaaccagggaaagttcctaagctcctggt ctttgctgcatccactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggacagatttcactct caccatcagcagcctgcagcctgaggatgttgcaacttattactgtcaaaggtataacagtgccccgctca 30 ctttcggcggagggacgaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccat ctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaa agtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggaca

gcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacaca

aagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga

gagtgt

Clone No 861:

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgg gcaagtcagatcattgccagctatttaaattggtatcagcagaaaccaggcagagcccctaagctcctgat ctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcact CTCA ca ccatcag g tctgca the CCTG a to g attttg the ctta ca CTA CAG ctgtca to the GTTA ca 10 gtacccccatat tcactttcggccctgggaccaaggtgaatatcaaacgaactgtggctgcaccatctgtcttcatcttcccgcc atctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcc aaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcagga cagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaaca caaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggg 15 gagagtgt

Clone No 863:

gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagg accagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctca tctatgatgcttccaatagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttca 20 ctctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagtgactggctcact ttcggcggagggaccaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatct gatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaa gtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacag caaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaa agtgt 25 agtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggag

Clone No 868:

gaaattgtaatgacacagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcag ggccagtcagagtattaaaaacaacttggcctggtaccaggtgaaacctggccaggctcccaggctcctc acctctggtgcatccgccagggccactggaattccaggcaggttcagtggcagtgggtctgggactgactt cactctcaccatcagcagcctccagtctgaagatattgcagtttattactgtcaggagtataataattggcccc tgctcactttcggcggagggaccaaggtggagatccaacgaactgtggctgcaccatctgtcttcatcttcc cgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagaga ggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagc aggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgaga 5 aacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaaca ggggagagtgt

Clone No 870:

gacatccagatgacccagtctcctccctccctgtctgcatctgtgggagacagagtcaccatcacttgccgg gcaagtcagaggattgccagctatttaaattggtatcagcagaaaccagggagagcccctaagctcctga 10 tctttgctgcatccagtttacaaagtggggtcccatcaaggttcagtggcagtggatctgggacagacttcac tctcaccatcagtagtctgcaacctgaagattatgcgacttactactgtcaacagagttacagtactcccatct acacttttggccaggggaccaagctggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgc catctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggc caaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcagg 15 acagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaac acaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagg ggagagtgt

Clone No 871:

20 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccag gcgagtcagggcattagcaactatttaaattggtatcaacagaaaccagggaaagcccctaagctcctga tcttcgatgcatccaatttggaatcagaggtcccatcaaggttcagtggacgtggatctgggacagattttact ttctccatcagcagcctgcagcctgaagatattgcaacatatttctgtcaacagtatgataatttcccgtacact tttggccaggggaccaagctggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatct gatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaa 25 gtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacag caaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaa agtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggag agtgt

Clone No 880:

gacatccagatgacccagtctccatcctccctggctgcatctgtaggagacagagtcaccatcacctgccg ggcaagtcagacgattgccagttatgtaaattggtatcaacagaaaccagggaaagcccctaatctcctg atctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttca ctctcaccatcagcagtctgcaacctgaagattttgcatcttacttctgtcaacagagttacagtttcccgtaca cttttggccaggggaccaagctggatatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatc tgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaa 5 gtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacag caaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaa agtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggag agtgt

Clone No 881:

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgg gcaagtcagaccattgccagctatgtaaattggtatcagcagaaaccagggaaagcccctaagctcctg atctatgctgcatccaatttgcaaagtggggtcccttcaaggttcagtggcagtggatctgggacagatttca ctctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtgtccctcg gctcactttcggcggagggaccaaggtggacatcacacgaactgtggctgcaccatctgtcttcatcttccc 15 gccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagag gccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagca ggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaa acacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacag gggagagtgt

Clone No 884:

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgg tcaagtcagaccattagcgtctttttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatc tatgccgcatccagtttgcacagtgcggtcccatcaaggttcagtggcagtggatctgggacagatttcactc tcaccatcagcagtctgcaacctgaagattctgcaacttactactgtcaagagagtttcagtagctcaactttc 25 ggcggagggaccaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctga tgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagta cagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagca aggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaag gt tctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagt 30 clone in 886:

gaaattgtaatgacacagtctccagccaccctgtctgtgtctccaggggaaacagccaccctctcctgcag ggccagtcagagtgttagcagcaacttagcctggtaccaacataaacctggccaggctcccaggctcctc atccatagtgcatccaccagggccactgggatcccagccaggttcagtggcagtgggtctgggacagagt 5 tcactctcaccataagcagcctgcagtctgaagattttgcagtttattactgtcagcagtataatatgtggcctc cctggacgttcggccaagggaccaaggtggaaatcaaacgaactgtggctgcaccatctgtcttcatcttc ccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagaga ggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagc aggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgaga 10 aacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaaca ggggagagtgt

Clone No 888:

gatattgtgatgacccagtctccactctccctgcccgtcacccctggagcgccggcctccatctcctgcaggt ctagtcagagcctcctgcgtactaatggatacaactatttggattggtacctgcagaagccagggcagtctc 15 cacagctcctgatctatttgggttctattcgggcctccggggtccctgacaggttcagtggcagtggctcagg cacagattttacactgaaaatcagcagagtggaggctgaggatgttggggtttattactgcatgcaatctcta caaacttcgatcaccttcggccaagggacacgactggagattaaacgaactgtggctgcaccatctgtctt catcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcc cagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtca 20 cagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagact acgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagag cttcaacaggggagagtgt

Clone No 894:

gaaattgtaatgacacagtctccagccaccctgtctgtgtctccgggggaaagagccaccctctcctgcag 25 ggctagtcagagtgttggcaacaacttagcctggtaccagcagagacctggccaggctcccagactcctc atctatggtgcgtccaccagggccactggtatcccagccaggttcagtggcagtgggtctgggacagagtt cactctcaccatcagcagcctgcagtctgaggattttgcagtttattactgtcagcagtatgataagtggcctg agacgttcggccaggggaccaaggtggacatcaagcgaactgtggctgcaccatctgtcttcatcttcccg ccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagagg 30 ccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcag gacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaa cacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagg

ggagagtgt

In all 44 clones discussed above, the encoded antibodies include the same constant IgG heavy chain, which has the following 5 amino acid sequence (SEQ ID NO: 178):

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGK 10 EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK

The genomic sequence encoding this heavy chain has the following nucleic acid sequence (SEQ ID NO: 177):

15 aqtacctccaccaaaaacccatcaatcttccccctaacaccctcctccaaaaacacctctaaaaacaca

taaccaacaacatacacaccttcccaactatcctacaatcctcaaaactctactccctcaacaacataata

accataccctccaacaacttaaacacccaaacctacatctacaacataaatcacaaacccaacaacac caaaataaacaaqaqaqttgqtgagagqccagcacagqqagggaqggtgtctgctgqaagccaggct (20 cagcgctcctgcctggacgcatcccggctatgcagtcccagtccagggcagcaaggcaggccccgtctg cctcttcacccggaggcctctgcccgccccactcatgctcagggagagggtcttctggctttttccccaggct ctgggcaggcacaggctaggtgcccctaacccaggccctgcacacaaaggggcaggtgctgggctca gacctgccaagagccatatccgggaggaccctgcccctgacctaagcccaccccaaaggccaaactct ccactccctcaqctcoqacaccttctctcctcccaaattccaataactcccaatcttctctctqcaoaqccca aatcttqtqacaaaactcacacatocccaccQtqcccaaqtaaqccaqcccaqqcctcqccctccaqctc aaggcgggacaggtgccctagagtagcctgcatccagggacaggccccagccgggtgctgacacgtc cacctccatctcttcctcaqcacctaaactcctqqqqqqaccqtcaqtcttcctcttccccccaaaacccaa qaacaccctcatoatctcccqqacccctaaggtcacatocqtoqtqgtggacgtqaqccacqaaoaccc taaqatcaaqttcaactqqtacataaacqocQtqaaqatacataatqccaaqacaaaaccacaaqaqq aqcaqtacaacagcacgtaccqtqtqqtcaqcqtcctcaccqtcctqcaccaqqactqqctqaatqgca aoqaqtacaaqtqcaaqqtctccaacaaaqccctcccaocccccatcqaqaaaaccatctccaaaqcc

aaaqqtqqqacccgtggggtgcqaqqqccacatqqacaqaggccggctcggcccaccctctgccctg agagtaaccgctataccaacctctatccctacaaaacaaccccaaaaaccacaaatatacaccctaccc ccatcccaaaaaaaaataaccaaaaaccaaatcaacctaacctacctaatcaaaaacttctatcccaac aacatcaccataaaataaaaaaacaataaacaaccaaaaaacaactacaaaaccacacctcccatac taaactccaacaactccttcttcctctataacaaactcaccataaacaaaaacaaataacaacaaaaaaa 5 catcttctcatactccataatacataaaactctacacaaccactacacacaaaaaaacctctccctatcccc aaataaataa

In this sequence, exons are indicated by double underscore. In addition, ser-encoding agt nucleotides (bold underlined) are created as a result of introduction into the XHoI-digested expression vector of an XHo-digested PCR product encoding the variable heavy chain site in the IgG expression vector.

The Vh and Vl coding pairs discussed above were selected according to the binding specificity for various ELISA and / or FLISA antigens and peptides, epitope mapping, 15 antigen diversity, and sequence diversity. The selected cognate V gene pairs were subjected to clone repair (Example 1, Section f) if errors were identified. Individual expression constructs were cotransfected with a Flp-recombinase expression palm in the CHO-FIpIn recipient cell line (Invotrogen), followed by selection of antibiotics from the members. Transfections, selection and adaptation to serum free culture were performed as described in Example 1, section g1 and g-2.

Stably transfected serum-free culture adapted to individual expression cell lines were cryopreserved in multiple ampoules to generate a cell bank of individual antibody-producing cell lines.

EXAMPLE 3

In vitro neutralization experiments were performed with both single antibody clones and purified antibody combinations. All antibody mixtures described below are made up of various anti-RSV antibodies of the present invention, which have been combined into one mixture using equal amounts of the different antibodies. Simple antibody test

Initially, the neutralizing activity of each antibody was determined on PRNT in the presence of complement against subtype A and subtype RSV strains as described in Example 1, section j-2.

EC50 values of various purified antibodies are shown in Table 8. Interestingly, although most anti-F antibodies individually exhibited virus neutralizing activity, no EC50 value can be determined for most antiRSV G protein antibodies. This could be interpreted as indicating that these antibodies are not able to neutralize the virus individually. However, subsequent refining of the assay also generated EC50 values for G-reactive clones. Blank fields indicate that the analysis has not yet been performed. ND indicates that an EC50 value cannot be determined in PRNT due to very low or absent neutralizing activity.

Table 8: EC50 values of anti-RSV FeG protein antibodies against RSV of AeB subtypes.

Clone Specificity EC50 Value (pg / mL) Long A2 18537 B1 793 G 2.52 0.09 800 F 0.15 0.16 810 F 0.04-0.06 0.02 0.02-0.14 0 , 29 816 G ND ND 818 F (1.86) 0.21 0.15 * 819 F 0.18 0.09 824 F 0.03 0.007 0.02 0.07 825 F 0.12 0.04 827 F 0.16 0.10 831 F 0.08 0.72 1.66 853 G (1.49) 0.14 0.13 * 855 G 6.35 ND EC50 value (pg / mL) 856 G ND ND 858 F ND 0.13 868 G ND 880 F 0.38 0.95 0.40 888 G 0.14 894 F 0.08 0.07 Syna-F 0.14 0.15 0.20 gis * new determination value Anti-F antibody mixtures

The ability of anti-RSV protein F antibody mixtures to neutralize RSV strains of AeB subtypes was compared with the neutralizing effect obtained using Palivizumab (also an anti-F antibody). Neutralization capacity was assessed using the microneutralization test or PRNT as described in Example 1, Section j. In an initial experiment two antibody mixtures, anti-F (1) and anti-F (ll), containing five and eleven distinct anti-F antibodies, respectively, were compared to Palivizumab using the microneutralization test. Anti-F (I) is composed of antibodies obtained from clones 810, 818, 819, 825 and 827. Antibodies 810e819se bind to the AJW antigen site, antibody 818 to site B / l or F1, antibody 825 binds to a complex epitope overlapping sites A and C and antibody 827 binds to another complex epitope overlapping site IV (see Table 6). Anti-F (II) is composed of antibodies obtained from clones 735, 800, 810, 818, 819, 825, 827, 863, 880, 884 and 894. Anti-F (II) contains multiple ligands for some of the antigenic sites. defined: antibodies 810, 819 and 863 bind to A / ll, antibodies 800 and 818 to F1 (or B / 1), antibodies 827 and 825 to the complex epitopes described above, antibodies 735 and 894 belong to to the unknown group (UC) I, antibody 880 to UCII, and antibody 844 binds to another currently unknown epitope (see Table 6). As shown in Figure 5, both Anti-F (I) and F (II) compositions were more potent than Palivizumab with respect to neutralizing RSV strains of both subtypes.

Figure 5 also shows that the combination of five antibodies (anti-F (1)) seemed more potent than the combination of eleven antibodies (AntiF (II)). The anti-F (1) mixture contains some of the most potent individually neutralizing antibodies of the specificities of different epitopes that have been defined so far. The anti-F (II) mixture contains the same five highly potent antibodies, but also contains additional ligands for some of the defined epitopes and the included antibodies also exhibit a broader range of neutralizing activity by itself. It is also possible that the activity of highly potent antibodies may become diluted in the anti-F (II) combination due to competition for binding to neutralizing epitopes on protein F. However, there are potentially already other effects than the neutralizing effect associated with each antibody. individual, eg increased phagocytosis, antibody-dependent cell cytotoxicity (ADCC), anti-inflammatory effects, complement activation, and a decreased likelihood of generating escape mutants, when considered in vivo, this result should not be taken as a Indication that a mixture of five is better than a mixture of eleven antibodies when used in vivo.

Both in vitro assays and clone combinations were

refined since this initial experiment and various combinations of F-specific antibody clones that are highly potent in the presence of complement have been identified. Neutralizing potencies, expressed as EC50 values (effective concentrations required to induce a 50% reduction in plaque count), of additional anti-F antibody composition are listed in Table 9. Regardless of the exact number of clones in the compositions , most of the F-specific antibody combinations tested are more potent than Palivizumab with respect to neutralization of the RSV subtype A strain.

Anti-G antibody mixtures

The ability of anti-G antibody mixtures to neutralize RSV subtype A strains was tested using PRNT as described in Example 1, section j-2. EC50 values of the tested anti-G antibody compositions are listed in Table 9. Most of the two anti-G antibody compositions did not exhibit markedly marked ability to neutralize viruses compared to individual anti-G antibodies. Some combinations of two or three anti-G antibodies never reached 100% neutralization of the virus, regardless of concentration. However, when additional anti-G antibodies were added to the composition, potency increased possibly indicating a synergistic neutralizing effect between anti-G antibodies. Figure 7 shows an example of increased potency when multiple G specific clones are combined.

Anti-F and anti-G antibody mixtures

The ability of the protein F and protein G anti-RSV antibody mixtures to neutralize the subtype B RSV strain was compared with the neutralizing effect obtained using Palivizumab. Neutralization capacity was assessed using both the microneutralization fusion inhibition assay as described in Example 1, section j-4 as well as the plate reduction neutralization assay (Example 1, section j-2).

Initially, the neutralizing activity of two antibody mixtures, anti-F (1) G and anti-F (ll) G, was measured in the microneutralization fusion inhibition assay. Each of these mixtures contains the anti-F antibodies of the anti-F (1) and anti-F (ll) composition described above as well as the anti-G antibodies obtained from clones 793, 796, 838, 841, 856 and 888, wherein antibodies 793, 796 and 838 bind to the conserved region of protein G, 841 and 856 bind to RSV GCRR of subtype A and 888 bind to GCRR of both subtypes (see Table 6). As shown in Figure 6, both Anti-F (I) G and F (II) G compositions were more potent than Palivizumab with respect to neutralization of RSV strain B1. Also, the neutralizing activity of the two mixtures was about the same. Thus, it appears that when anti-F antibodies are combined with several specific G protein clones, the power difference previously observed between the two anti-F antibody mixes is diminished. This may indicate a general increase in neutralizing activity when antibodies that recognize a broad range of RSV antigens and epitopes are combined.

A large number of different combinations of both anti-F and anti-G antibodies have since been tested on PRNT in the presence or absence of complement. EC50 values obtained by this assay in the presence of active complement are shown in Table 9. All compositions tested including both anti-F and anti-G antibodies actually neutralized subtype A RSV and most of these are more potent than Palivizumab.

Results and results shown in Tables 7 and 8 mos

They also find that antibodies with naturally high affinities could be repeatedly obtained from human donors using the antibody cloning technique of the present invention.

Table 9: EC50 values of the anti-RSV antibody combinations against subtype A and subtype B. Blank fields indicate that the analysis has not yet been performed. ND indicates that an EC5O value cannot be determined in PRNT due to very low neutralization activity or absence thereof.

Antibody Number in Composition EC50 Value (pg / mL) Long A2 18537 B1 1 810, 818, 819,825, 827 0.19 0.38 2 810, 818, 819, 825, 827, 831, 858, 0.34 863 , 884, 894, 793, 796, 816, 838, 853, 856, 859, 888, 3 810, 818, 825, 827, 884, 886, 793, 0.30 853, 868, 888 4 810, 818, 825, 827, 831, 858, 884, 0.19 886, 793, 796, 816, 853, 856, 868, 888 810, 818, 825, 827, 831, 858, 884, 0.21 886, 793, 853, 868 , 888 6 810, 819, 825, 827, 831, 793, 853, 0.20 856, 858, 868 7 810, 811, 817, 819, 825, 827, 831, 0.18 838, 853, 856, 858 , 859, 863, 868 EC50 Value Number (pg / mL) Antibodies in the composition Long A2 18537 B1 8 800, 801, 811, 838, 853, 855, 859, (ND) 861, 880, 894, 736, 795 , 796, 799 0.92 * 9 810, 818, 825 0.14 0.03 0.29 0.10 810, 818, 819, 825, 827, 884 0.21 0.42 11 810, 818, 819, 825, 827, 884, 886 0.15 0.29 12 793, 816, 853, 856 0.06 13 793, 816, 853, 855, 856 0.03 0.0 0.86 3 14 793, 868, 888 , 853, 856 0.34 793, 796, 818, 816, 838, 853, 855, 0.11 856, 859, 868, 888 16 810, 818, 827 0.11 0.21 17 810, 818, 825, 827, 858, 886 0.10 0.05 0.16 18 810, 818, 825, 827, 858, 886, 793, 0.04 0.06 0.15 816, 853, 855, 856 19 818, 825, 827, 858, 886, 793, 816, 0.06 853, 855, 856 810, 818, 819, 825, 827, 858, 793, 0.10 0.06 816, 853, 855, 856 21 810, 793, 816, 853, 855, 856 0.04 22 818, 825, 827, 831, 858, 886, 793, 0.06 816, 853, 855, 856 23 818, 825, 827, 831, 858, 819, 793, 0.06 0.03 816, 853, 855, 856 24 818, 827, 831, 858, 819, 793, 816, 0.06 0.04 853, 855, 856 810, 818, 819, 824, 825, 827 , 858, 0.07 793, 816, 853, 855, 856 26 831, 818, 819, 824, 8 25, 827, 858, 0.08 793, 816, 853, 855, 856 27 831, 818, 819, 824, 827, 858, 793, 0.05 816, 853, 855, 856 28 810, 818, 824 0 0.03 0.04 0.04 0.04 0.06 4 29 810, 824 0.05 818, 824 0.04 Antibody number in the composition EC50 value (pg / ml) Long A2 18537 B1 31 810.818 0, 08-0.11 32 824, 793, 816, 853, 855, 856 0.05 33 810, 818, 819, 824, 825, 827, 858, 0.03-0.03 0.06 894, 793, 816, 853, 855, 856 0.07 6 34 810, 818, 819, 824, 825, 827, 894, 0.07 793, 816, 853, 855, 856 793,816 5.94 36 855,856 ND 37 793, 856 ND 38 793, 853 2.35 39 853, 856 0.21 40 793, 853, 856 2.84 41 793, 816, 853 1.97 42 853, 855, 856 0.25 43 793, 816, 853, 856 0.45 44 793, 853, 855 0.26 45 793, 853, 855, 856 0.16 46 816, 853, 855, 856 0, 07 47 816, 856 0.06 48 816, 853 0.75 49 816, 853, 856 0.07 50 810, 818, 824, 816 0.09 51 810, 818, 824, 853 0.11 52 810, 818,824,856 0.10 53 810, 818,824,816,853 0.09 54 810, 818, 824, 816, 856 0.05 55 810, 818, 824, 853, 856 0.08 56 810, 818,824,816,853, 856 0.05 0.0 0, 03 0.06 3- 0.0 5 Palivizumab (Synagis) 0.14 0.1 0.20 5 * new determination value Example 4

Reduction of viral loads in the lungs of RSV-infected mice.

The in vivo protective ability of purified antibody combinations of the invention against RSV infection has been demonstrated in the BALB / c mouse model (Taylor et al. 1984. Immunoiogy 52, 137-142; Mejias, et al. 2005. Antimicrob. Agents Chemother 49: 4700-4707) as described in Example 1, Section k-1. Table 10, data from an experiment with three different anti-RSV rpAb consisting of equal amounts of different antibody clones of the invention (described in Table 9), are presented in comparison to data from uninfected control animals and treated animals. with placebo (PBS) from the same experiment. Each treatment group contained 5 mice and samples were obtained on the fifth day after infection, which is approximately at the peak of viral replication in this model. As shown in Table 10, rpAb combinations effectively reduce viral load by at least one order of magnitude when administered prophylactically. Copy numbers are presented as means ± standard deviation. The number of copies was below or within the detection limit of this assay, ie 3.8 10 8 RNA copies / mL, for two of the treatment groups.

Table 10: Viral load in the lungs of mice after pro

phylaxis and the challenge with RSV.

Treatment group RT-PCR viral load New data (Iog10 RNA copies / ml) Uninfected Neqativo PBS 5.14 ± 0.09 4.25 rpAb anti-RSV 18 (50 mq / kq) ND rpAb anti-RSV 18 (5 mq / kq) 4.61 ± 0.22 3.64 rpAb anti-RSV 9 (50 mq / kq) ND rpAb anti-RSV 9 (5 mq / kq) Small 4.74 ± 0.38 3.82 rpAb anti-RSV 17 (50 mq / kq) kq) 4.41 ± 0.14 3.04 rpAb anti-RSV 17 (5 mq / kq) Lar- 4.69 ± 0.05 3.90 The samples were then analyzed using an RT-PCR configuration. different quantitative (described in section k-1). In Table 11a, data from an experiment with four different anti-RSV rpAb consisting of equal amounts of different antibody clones of the invention (described in Table 9) and clone 810 alone are presented compared to data from non-control animals. infected animals and placebo-treated animals (PBS) from the same experiment. Each treatment group contained 5 mice and samples were obtained on the fifth day after infection, which is approximately at the peak of viral replication in this model. As shown in Table 11a, rpAb combinations effectively reduce viral load by at least one order of magnitude 10 when administered prophylactically to 25 mg / kg body weight. Copy numbers are presented as means ± standard deviation.

Table 11a: Viral burden of mouse lungs after pro

phylaxis and challenge with RSV.

Treatment Group (dose) RT-PCR viral load (Iog10 RNA copies / ml) Uninfected Neqativa PBS 4.11 ± 0.12 rpAb anti-RSV 18 (25 mq / kq) 2.74 ± 0.16 rpAb anti RSV 18 (5 mq / kq) 3.40 ± 0.09 rpAb anti-RSV 9 (25 mq / kq) 2.95 ± 0.19 rpAb anti-RSV 9 (5 mq / kq) 3.56 ± 0.31 roAb anti-RSV 17 ( 25 mq / kq) 2.81 ± 0.29 rpAb anti-RSV 13 (5 mq / kq) 3.39 ± 0.12 rpAb anti-RSV 13 (25 mq / kq) 3.02 ± 0.33 rpAb anti-RSV 13 ( 5 mq / kq) 3.34 ± 0.26 Clone 810 (25 mq / kq) 3.03 ± 0.16 Clone 810 (5 mq / kq) 3.37 ± 0.22 In Table 11b, data from a second study with three rpAb an

Different ti-RSVs, consisting of equal amounts of different antibody clones of the invention (described in Table 9) and clone 824 alone are presented compared to data from uninfected control animals and placebo treated animals (PBS) and animals treated with 20 Palivizumab (Synagsis) from the same experiment. Each treatment group contained 5 mice and samples were obtained on the fifth day after infection. Copy numbers are presented as means ± standard deviation. In Table 11c, data from a third anti-RSV 33 rpAb study consisting of equal amounts of different antibody clones of the invention (described in Table 9) and clone 824 alone are presented compared to data from non-control animals. infected and placebo treated animals (PBS) and Palivizumab treated animals (Synagsis) from the same experiment. Each treatment group except the last one contained 5 mice and samples were obtained on the fifth day after infection. One mouse was removed from each rpAb anti-RSV 33 treated group at

15, 5 and 1.5 mg / kg body weight, as it was found that they never had the antibody injected. Copy numbers are presented as means ± standard deviation.

In all three studies, there was a statistically significant reduction in RSV RNA copy number in antibody-treated groups compared to placebo-treated control (p <0.05; homoscedastic t-test). In the second study, viral load in the antibody-treated groups of the invention is significantly lower than that of the corresponding Synagis-treated groups (Table 11b). In the third study, viral load is significantly lower in rpAb anti-RSV 33-treated groups than in Synagis-treated groups at all doses tested (Table 11 c).

Table 11b: Viral loads in the lungs of mice after

prophylaxis and challenge with RSV.

Treatment Group (dose) RT-PCR viral load (Iog10 copies of RNA / ng total RNA) Uninfected Neqativa PBS 4.22 ± 0.20 Svnaqis (15 mq / kq) 3.68 ± 0.25 Svnaqis (3 mq / kq) 3.83 ± 0.12 rpAb anti-RSV 28 (15 mq / kq) 2.96 ± 0.19 rpAb anti-RSV 28 (3 mq / kq) 3.32 ± 0.23 rpAb anti-RSV 33 (15 mq / kq) 2.9510.30 rpAb anti-RSV 33 (3 mq / kq) 3.66 + 0.07 rpAb anti-RSV 56 (15 mq / kq) 2.6610.18 rpAb anti-RSV 56 (3 mq / kq) 3.25 + 0.38 Clone 824 (15 mq) / kq) 2.51 + 0.28 Clone 824 (3 mq / kq) 3.09 + 0.18 Table 11c: Viral loads in the lungs of mice after

prophylaxis and challenge with RSV. The asterisks indicate that the groups contained only four animals. _

Treatment Group (dose) RT-PCR viral load (Iog10 RNA copies / ng total RNA) Uninfected Negative PBS 4.13 ± 0.17 Synagis (45 mg / kg) 3.56 ± 0.22 Synagis (15 mg / kg) 3.60 ± 0.27 Synagis (5 mg / kg) 3.77 ± 0.14 Synagis (1.5 mg / kg) 3.86 ± 0.12 rpAb anti-RSV 33 (45 mg / kg) kg) 2.38 ± 0.18 rpAb anti-RSV 33 (15 mg / kg) * 2.70 ± 0.18 rpAb anti-RSV 33 (5 mg / kg) * 3.15 ± 0.24 rpAb anti-RSV 33 RSV 33 (1.5 mg / kg) * 3.53 ± 0.12 Finally, Table 11d, data from a long-term study

anti-RSV 33 rpAb, consisting of equal amounts of different antibody clones constituting rpAB 33 (described in Table 9), are presented in comparison to data from uninfected control animals and placebo-treated animals (PBS) from the same experiment. . Each treatment group contained 5 mice and samples were obtained on days 5, 10 27 and 69 after infection. Copy numbers are presented as means ± standard deviation. Due to very low copy numbers on day 69 post infection, the copy number was calculated per mL of lung tissue. The detection limit of this assay is approximately 2 10 10 RNA copies / ml of homogenized lung tissue.

At all three time points, there is a statistical reduction

significantly higher RSV RNA copy number in antibody-treated groups compared to placebo-treated control (p <0.01; homoscedastic t-test) (Table 11 d).

Table 11 d: Viral loads in mouse lungs after RSV prophylaxis and challenge. Each treatment group contained 5 animals per time point. Treatment Group (RT-PCR viral load if) (Iog10 RNA copies / mL of homogenized lung) Day 5 Day 27 Day 69 Uninfected Negative Negative Negative PBS 9.36 ± 0.15 4.52 ± 0.22 4.05 ± 0.18 rpAb 33 anti-RSV (45 7.51 ± 0.22 3.22 ± 0.22 2.68 ± 0.41 mg / kg) Cytokine and chemokine levels in samples pulmonary

RSV-infected mice.

Lung samples from a pilot study of prophylaxis in ca5 mice were analyzed by a commercial multiplex immunoassay to determine the levels of different cytokines and chemokines following RSV infection and antibody prophylaxis with rpAb 18 (Table 9) as described. in Example 1, Section k-1. Samples of uninfected and untreated animals were also analyzed to obtain normative data for the BALB / c mouse. All samples were obtained on the fifth day after infection. Data are presented as means ± standard deviation.

The analysis showed (Table 12) that the levels of some cytokines and chemokines that were indicated to be important markers of an RSV infection and the subsequent inflammatory response in both humans and mice, including interferon (IFN) -y, interIeucine. (IL) -I β, IL-4, IL-6, IL-8 (KC / GROa), IL-10, macrophage inflammatory protein (MIP) -Ia, regulated by activation of expressed and secreted normal T cells (RANTES, CCL5) and tumor necrosis factor (TNF) -α were elevated in the lungs of placebo-treated animals, while lungs treated with approximately 50 mg / kg rpAb had levels about the same as those of non-control animals. -infected. Similar results were also obtained with other anti-RSV rpAb combinations. It should be noted that mice do not have a clear cut homologue for IL-8, but they do have a functional homologue for human GROa (similar in function to IL-8) called KC. The kinetics of the inflammatory response and dose response effects of antibody prophylaxis remain to be investigated.

Table 12: Cytokine and chemokine levels in lung tissue

mouse mice infected with RSV. Sample level of mice Tra¬ mice Tra¬ mice Non-infected placebo-treated rpAb mice (pg / mL) anti-RSV control IL--β 270 ± 71 570 + 100 310 + 140 IL-4 7.7 ± 4.7 26 + 4.6 14 + 8.5 IL-6 6.4 ± 2.6 22112 8.213,8 IL-10 120 ± 17 320 + 58 170141 IFN-γ 20 ± 7.6 420188 81172 KC / GROa (IL-8) 51138 290 + 83 94 + 99 MIP-Ia (CCL3) 39 + 16 9401170. 160 + 110 RANTES (CCL5) 60 + 28 380 + 32 140 + 66 TNF-a 18 + 6.1 95110 38 + 25 Effect of antibody prophylaxis on pulmonary and infile pathology

cell traction in RSV-infected mice.

Histopathological lung tissue samples from the long-term studies were examined for signs of inflammation and scoring classified according to the system described in Example 1, Section k10 1. Each treatment group contained 5 mice and samples were obtained within days. 5, 27 and 69 after infection. One mouse was removed from the group and treated with anti-RSV 33 rpAb and killed on the fifth day after infection as antibodies were found not to be injected into it. Data are presented as means ± standard deviation. Five days after RSV infection 15, there was a significant increase in lung pathology in placebo-treated mice compared to uninfected control mice (p <0.05; homeoscedastic t-test; table 13). Signs of inflammation decreased over time, but were still significant compared to uninfected control mice on day 69 after infection (p <0.0001). Pulmonary pathology was characterized by peribronchiolar and perivascular accumulations of lymphocytes and alveolitis lymphocytes on days 27 and 69 after infection. On the other hand, there was little or no lymphoid accumulation in the lungs of antibody-treated mice and the lungs were similar to those of uninfected control mice. Pulmonary pathology in placebo-treated mice was also significantly higher than in antibody-treated mice (p <0.02 on day 5, <0.03 on day 27 and <0.0001 on day 69).

Table 13: Mouse Pathology Score

after prophylaxis and RSV challenge. The asterisk indicates groups containing only four animals._

Treatment group (do¬ Pulmonary pathology score (Grase) age x Prevalence) Day 5 Day 27 Day 69 Uninfected 0 0.4 ± 0.5 0 PBS 10.8 ± 4.3 2.8 ± 1, 1 2.6 ± 0.5 rpAb 33 anti-RSV (45 mg / kg) 3 ± 0.8 * 0.6 ± 0.9 0.2 ± 0.4 10

Perivascular and peribronchiolar lymphoid accumulations in the lungs of RSV-infected mice 28 and 70 days after infection correlated with increased BAL lymphocyte values. As shown in Table 14, there was a significant increase in the number of inflammatory cells in BAL 15 of placebo-treated mice compared to uninfected control mice on days 5, 27, and 69 post-infection (p <0.002; p <0 , 03 and p <0.03, respectively). Pulmonary inflammatory response in mice treated with anti-RSV 33 rpAb was significantly lower than that of placebo 20 treated mice at all time points (p <0.003 on day 5; p <0.03 on day 27; p <0, 02 on day 69, respectively). The groups are the same as the pulmonary pathology described above and the data are presented as means ± standard deviation.

Table 14: BAL leukocyte count after prophylaxis and RSV challenge. The asterisks indicate groups that contained only four animals. Treatment group (dose) BAL cell count (x105) Day 5 Day 27 Day 69 Uninfected 1.1 ± 0.2 1.4 ± 0.3 1.0 ± 0.1 PBS 7.2 ± 2 3.0 ± 1.0 1.8 ± 0.5 rpAb anti-RSV 33 (45 mg / kg) 1.8 ± 0.5 * 1.5 ± 0.3 0.9 ± 0.1 Pharmacokinetics of human rpAb in mice.

The pharmacokinetic profile of the purified antibody combinations of the invention was determined in BALB / c mice as described in Example 1, Section I. Two different anti-RSV 33 rpAb and anti-RSV 56 rpAb (see Table 9), consisting of equal amounts of different antibody clones of the invention were investigated. Each treatment group consisted of 15 mice. Human IgG levels in serum and lung homogenate samples correspond to the level of anti-RSV 10 rpAb present at specific time points.

Figure 8 shows serum pharmacokinetic profiles of rpAb anti-RSV 33 and rpAb anti-RSV 56 at an antibody dose of 15 mg / kg. Using these data, some parameters were determined, which are summarized in Table 15. The two different anti-RSV 15 rpAb compositions have similar 11-day half-life (T1 / 2) pharmacokinetic profiles. These findings were verified using a dosage of 37.5 mg / kg.

Table 15. Pharmacokinetic data of serum obtained in mice.

dongos.

Cmax Tmax (hours) AUC (0-694) * Ty2 (days) (mg / ml) anti-RSV rpAb rpAb 33 15.4 25 51 11.1 anti-RSV rpAb 56 17.2 25 52 11.0 * Area under the curve between time = 0 hours and time = 694 hours.

As the primary target of RSV during infection is lung tissue, the presence of anti-RSV rpAb in this tissue is vital for efficacy. To determine the distribution of anti-RSV 33 rpAb and anti-RSV 56 rpAb (according to Table 9) in lung tissue, IgG levels were measured in lung homogenates on days 1 (25 hours), 6 and 29 after antibody treatment. At all time points measured, rpAb anti-RSV 33 and rpAb anti-RSV 56 levels in lung tissue were found to be nearly identical to the maximum mean IgGI concentration, about 0.006 mg / mL on the first day after treatment. By days 6 and 29, the levels had decreased. However, IgGI could still be measured in lung tissue on day 29 after antibody treatment. These findings show not only that the anti-RSV 33 rpAb and anti-RSV 56 rpAb compositions are present shortly after treatment, but also that antibodies can be found at detectable levels up to 29 days after treatment.

Claims (36)

1. Anti-RSV antibody capable of competing in binding to antibody 824 as defined herein, or with a Fab fragment based thereon. An anti-RSV antibody comprising a CDRH3 of formula: wherein X 1 to X 5 are individually selected from the amino acid groups listed below: X I = R or K; X 2 = D, E, N or Q; X 3 = S, T, G or A; X4 = S, T, G or A; X5 = N, Q, D or E; X6 = W, Y, F or H; X7 = A, G, V, or S; X8 = G, A, V, or S; X9 = Y, F, W or H; X10 = E or D; and XII = D, E, N or Q; and a CDRL3 described by the following formula: wherein X1 to X7 are individually selected from the amino acid groups listed below: X1 = Q or H; X 2 = Q, E or H; X 3 = F, Y, W or H; X 4 = N 1 Q or H; X5 = T1 S, G or A; X6 = Y, F1 W or H; and X7 = F1 Y1 W or H. Antibody according to claim 2, comprising the CDR1 and CDR2 regions of the Vh and Vl pair of antibody 824 as set forth in SEQ ID NOs: 232, 317, 487, and 572, wherein up to 2 amino acids have been exchanged. compared to said SEQ ID NOs. The antibody of claim 2, comprising the CDR3 region of formula CARDSSNWPAGYEDW (SEQ ID NO: 402), and a CDRL3 region of formula CQQFNTYPFTF (SEQ ID NO: 657). The antibody of claim 2, comprising the Vh region (SEQ ID NO: 9) of antibody 824. The antibody of claim 2, comprising the V1 region (amino acids 1 to 107 of SEQ ID NO: 107) of antibody 824. The antibody of claim 2, comprising the light chain (SEQ ID NO: 107) of antibody 824. An antibody according to claim 2, comprising Ch as defined in SEQ ID NO: 178. Antibody according to claim 2, having the binding specificity of antibody 824. Antibody according to claim 1 or 2, capable of neutralizing RSV subtypes A and B in a viral neutralization assay. Antibody according to claim 1 or 2, capable of providing a significant reduction in RSV virus burden in the lungs of an RSV infected mammal. An antibody composition comprising antibodies as defined in any one of claims 1 to 11, and one or more additional anti-RSV antibodies. An antibody composition according to claim 12, wherein the additional anti-RSV antibodies are chosen from the group consisting of human antibodies, humanized antibodies and chimeric human mouse antibodies. An antibody composition according to claim 12, wherein the additional anti-RSV antibodies are chosen from the group consisting of antibody molecules described herein in Table 6, or a specifically binding fragment of such antibody molecule or an antibody. synthetic or semisynthetic analogue, such analog or ligand fragment comprising at least one of the complementarity determining regions (CDRs) of such an isolated antibody molecule, except for an antibody containing clone 824 CDRs. 15. Antibody composition, comprising distinct members comprising light and heavy chain CDR1, CDR2 and CDR3 regions selected from the group of Vh and Vl pairs listed in Table 6, wherein the distinct members are the distinct members of one of the composition. antibodies 2 to 56 in Table 9 here. An antibody composition according to claim 15 capable of neutralizing a subtype A RSV in a viral neutralization assay. An antibody composition according to claim 15 capable of neutralizing a subtype B RSV in a viral neutralization assay. The antibody composition of claim 15, wherein the distinct members are the distinct members of one of antibody compositions 2, 9,13,17, 18, 28, 33 and 56 of Table 9. A method for preventing, treating or alleviating one or more symptoms associated with an RSV infection in a mammal, comprising administering an effective dose of an anti-RSV antibody as defined in any one of claims 1 to 11, or an antibody composition. as defined in any one of claims 12 to 18 to such a mammal. The method of claim 19, wherein an effective amount is between 0.1 and 50 mg antibody per kg body weight. A method according to claim 19 or 20, wherein the antibodies are administered at least once a year. The method according to claim 20, wherein the antibodies are administered at regular intervals during the time of year when there is a greater risk of attracting an RSV infection. A method according to claim 21, wherein the regular intervals are weekly, biweekly, monthly or bimonthly. Use of a recombinant anti-RSV polyclonal antibody as defined in any one of claims 1 to 11, or antibody composition as defined in any one of claims 12 to 18 to prepare a composition for treating, alleviating or preventing one or more symptoms. associated with RSV infection in a mammal. An isolated nucleic acid fragment encoding the amino acid sequence of at least one CDR according to any one of claims 2 to 9. 26. Isolated nucleic acid fragment encoding the CDR sequences of a heavy chain amino acid sequence set forth in SEQ ID NO: 19. 27. Isolated nucleic acid fragment encoding the CDR sequences of a heavy chain amino acid sequence set forth in SEQ ID NO: 107. 28. Isolated nucleic acid fragment encoding the CDR sequences of a heavy chain amino acid sequence set forth in SEQ ID NO: 19 and the accompanying heavy chain amino acid sequences having SEQ ID NO: 107. A nucleic acid fragment according to any one of claims 25 to 28 which includes encoding the sequences comprised in SEQ ID NO: 63 and / or 151. A vector comprising the nucleic acid fragment as defined in any one of claims 25 to 29. The vector of claim 30 capable of autonomous replication. The vector of claim 30 or 31 selected from the group consisting of a plasmid, a phage, a cosmid, a minichromosome, and a virus. The vector of any one of claims 30 to 32, comprising, in the 5 '-> 3' direction and in the operative ligament, at least one promoter for triggering expression of a first nucleic acid fragment as defined in any one. one of claims 25 to 29, encoding at least one light chain CDR together with the necessary framework regions, optionally a nucleic acid sequence encoding constant regions and optionally a nucleic acid sequence encoding a first terminator, and / or - in the 5 '-' 3 'direction and in the operative ligament, at least one promoter for triggering expression of a second nucleic acid fragment as defined in any one of claims 25 to 29, which encodes at least one stranded CDR. along with the necessary framework regions, optionally a nucleic acid sequence encoding a leader peptide, said nucleic acid fragment, optionally has a nucleic acid sequence encoding constant regions and optionally a nucleic acid sequence encoding a second terminator. The vector of any one of claims 30 to 33 which, when introduced into a host cell, integrates into the host cell genome. Transformed vector-bearing cell as defined in any one of claims 30 to 34. Stable cell line which carries the vector as defined in any one of claims 30 and 34 and which expresses a nucleic acid fragment as defined in any one of claims 25 to 29 and which optionally secretes or carries its product. of recombinant expression on its surface.